Robert Andrews Millikan was born on March 22, 1868, in Morrison, Illinois. Millikan went to high school in Maquoketa, Iowa. Millikan received a bachelor's degree in the classics from Oberlin College in 1891 and his doctorate in physics from Columbia University in 1895 – he was the first to earn a Ph.D. from that department.

Millikan graduated from Oberlin College in 1891 and obtained his doctorate at Columbia University in 1895. In 1896 he became an assistant at the University of Chicago, where he became a full professor in 1910. In 1909 Millikan began a series of experiments to determine the electric charge carried by a single electron. He began by measuring the course of charged water droplets in an electric field. The results suggested that the charge on the droplets is a multiple of the elementary electric charge, but the experiment was not accurate enough to be convincing. He obtained more precise results in 1910 with his famous oil-drop experiment in which he replaced water (which tended to evaporate too quickly) with oil.

Although at the time of Millikan's oil-drop experiments it was becoming clear that there exist such things as subatomic particles, not everyone was convinced. Experimenting with cathode rays in 1897, J. J. Thomson had discovered negatively charged 'corpuscles', as he called them, with a charge-to-mass ratio 1840 times that of a hydrogen ion. Similar results had been found by George FitzGerald and Walter Kaufmann. Most of what was then known about electricity and magnetism, however, could be explained on the basis that charge is a continuous variable; in much the same way that many of the properties of light can be explained by treating it as a continuous wave rather than as a stream of photons.

In 1902 he married Greta Ervin Blanchard (1876-1955). They had three sons: Clark Blanchard, Glenn Allan, and Max Franklin.

When Einstein published his seminal 1905 paper on the particle theory of light, Millikan was convinced that it had to be wrong, because of the vast body of evidence that had already shown that light was a wave. He undertook a decade-long experimental program to test Einstein's theory, which required building what he described as "a machine shop in vacuo" in order to prepare the very clean metal surface of the photo electrode. His results published in 1914 confirmed Einstein's predictions in every detail, but Millikan was not convinced of Einstein's interpretation, and as late as 1916 he wrote, "Einstein's photoelectric equation... cannot in my judgment be looked upon at present as resting upon any sort of a satisfactory theoretical foundation," even though "it actually represents very accurately the behavior" of the photoelectric effect. In his 1950 autobiography, however, he simply declared that his work "scarcely permits of any other interpretation than that which Einstein had originally suggested, namely that of the semi-corpuscular or photon theory of light itself".

Starting in 1908, while a professor at the University of Chicago, Millikan worked on an oil-drop experiment in which he measured the charge on a single electron. J. J. Thomson had already discovered the charge-to-mass ratio of the electron. However, the actual charge and mass values were unknown. Therefore, if one of these two values were to be discovered, the other could easily be calculated. Millikan and his then graduate student Harvey Fletcher used the oil-drop experiment to measure the charge of the electron (as well as the electron mass, and Avogadro's number, since their relation to the electron charge was known).

In 1914 Millikan took up with similar skill the experimental verification of the equation introduced by Albert Einstein in 1905 to describe the photoelectric effect. He used this same research to obtain an accurate value of Planck’s constant. In 1921 Millikan left the University of Chicago to become Director of the Norman Bridge Laboratory of Physics at the California Institute of Technology (Caltech) in Pasadena, California. There he undertook a major study of the radiation that the Physicist Victor Hess had detected coming from outer space. Millikan proved that this radiation is indeed of extraterrestrial origin, and he named it "cosmic rays." As chairman of the Executive Council of Caltech (the school's governing body at the time) from 1921 until his retirement in 1945, Millikan helped to turn the school into one of the leading research institutions in the United States. He also served on the board of trustees for Science Service, now known as Society for Science & the Public, from 1921 to 1953.

In 1917, solar Astronomer George Ellery Hale convinced Millikan to begin spending several months each year at the Throop College of Technology, a small academic institution in Pasadena, California, that Hale wished to transform into a major center for scientific research and education. A few years later Throop College became the California Institute of Technology (Caltech), and Millikan left the University of Chicago in order to become Caltech's "chairman of the executive council" (effectively its president). Millikan would serve in that position from 1921 to 1945. At Caltech most of his scientific research focused on the study of "cosmic rays" (a term which he coined). In the 1930s he entered into a debate with Arthur Compton over whether cosmic rays were composed of high-energy photons (Millikan's view) or charged particles (Compton's view). Millikan thought his cosmic ray photons were the "birth cries" of new atoms continually being created to counteract entropy and prevent the heat death of the universe. Compton would eventually be proven right by the observation that cosmic rays are deflected by the Earth's magnetic field (and so must be charged particles).

Professor Millikan took sole credit, in return for Harvey Fletcher claiming full authorship on a related result for his dissertation. Millikan went on to win the 1923 Nobel Prize for Physics, in part for this work, and Fletcher kept the agreement a secret until his death. After a publication on his first results in 1910, contradictory observations by Felix Ehrenhaft started a controversy between the two physicists. After improving his setup, Millikan published his seminal study in 1913.

A religious man and the son of a minister, in his later life Millikan argued strongly for a complementary relationship between Christian faith and science. He dealt with this in his Terry Lectures at Yale in 1926–27, published as Evolution in Science and Religion. A more controversial belief of his was eugenics. This led to his association with the Human Betterment Foundation and his praising of San Marino, California, for being "the westernmost outpost of Nordic civilization ... [with] a population which is twice as Anglo-Saxon as that existing in New York, Chicago, or any of the great cities of this country."

Since Millikan's work formed some of the basis for modern particle physics, it is ironic that he was rather conservative in his opinions about 20th century developments in physics, as in the case of the photon theory. Another Example is that his textbook, as late as the 1927 version, unambiguously states the existence of the ether, and mentions Einstein's theory of relativity only in a noncommittal note at the end of the caption under Einstein's portrait, stating as the last in a list of accomplishments that he was "author of the special theory of relativity in 1905 and of the general theory of relativity in 1914, both of which have had great success in explaining otherwise unexplained phenomena and in predicting new ones."

In 1938, he wrote a short passage to be placed in the Westinghouse Time Capsules.

Robert Millikan was Vice Chairman of the National Research Council during World War I. During that time, he helped to develop anti-submarine and meteorological devices. He received the Chinese Order of Jade. In his private life, Millikan was an enthusiastic tennis player. He was married and had three sons, the eldest of whom, Clark B. Millikan, became a prominent aerodynamic Engineer. Another son, Glenn, also a Physicist, married the daughter (Clare) of George Leigh Mallory of "Because it's there" Mount Everest fame. Glenn was killed in a climbing accident in Cumberland Mountains in 1947.

Millikan died of a heart attack at his home in San Marino, California in 1953 at age 85, and was interred in the "Court of Honor" at Forest Lawn Memorial Park Cemetery in Glendale, California.

On January 26, 1982, he was honored by the United States Postal Service with a 37¢ Great Americans series (1980–2000) postage stamp.