In 2004, while hanging around in New York City's Lower East Side club scene, Ashford met nightlife personality Lady Starlight, a local rock DJ and performance Artist. Lady Starlight invited Ashford to dance at her 1970s glitter rock party "Lady Starlight's English Disco", and christened her Hollywood Starr. She performed in Tom Kitt and Brian Yorkey's Feeling Electric as Natalie at the New York Musical Theatre Festival in 2005.

Soon after graduating from college, Ashford got her big break performing as one of Glinda's friends, Pfanne, and understudying the role of Glinda on the First National Tour of Wicked. She joined the cast in December 2005 and left in September 2006. She then originated the role of Margot in Legally Blonde: The Musical, her Broadway debut. She also understudied the lead role of Elle Woods, but only went on once. She was featured on the original cast recording and appeared in the MTV televised airing. She left the show in September 2007.

She then returned to Wicked, this time playing the lead role of Glinda in the Broadway production. She replaced Kendra Kassebaum on October 9, 2007. She departed the company on May 11, 2008 when Kassebaum returned to the role, and transferred to the Chicago production, replacing Kate Fahrner as Glinda on June 3, 2008. She played her final performance as Glinda on January 25, 2009, when the Chicago production closed.

On April 7, 2008, Ashford made her professional cabaret debut as a part of the Broadway at Birdland series at Birdland Jazz Club. She performed an early version of what has now become her critically acclaimed Lost in the Stars show. Many songs from her debut, including Love Hurts, Mona Lisa S And Mad Hatters, and the titular song, Lost in the Stars, stayed with the show and are now featured on her live album. Alex Lacamoire served as her musical Director.

In 2009 it was announced that Ashford would take place as one of the principal characters and Singers in the new vaudeville styled Cirque du Soleil show, Banana Shpeel. However, it was announced that Ashford and her colleague, Michael Longoria, were no longer in the cast of the show. Publicists announced that it was a creative decision to remove large portions of dialogue from the show, therefore making their characters non-existent. Ashford was originally slated to stay in the production from its premiere in Chicago until sometime in 2010.

In November 2010, Ashford was part of a reading of the musical Carrie. In December 2010, she participated in a "creative workshop" reading of the stage musical version of Diner, as Beth.

In January 2012, Ashford ended her run playing Maureen Johnson in the Off-Broadway revival of Rent, which began July 14, 2011 and closed on September 9, 2012. Emma Hunton took over the role of Maureen from her on January 13, 2012. In February 2012, Ashford made a cameo on Smash as Lisa McMann, and returned for another short appearance in 2013 before cancellation.

From June 27 to August 19, 2012, she appeared in the Off-Broadway musical Dogfight, based on the 1991 film of the same name, playing a toothless hooker named Marcy. The role in the film was originally played by Elizabeth Daily.

Ashford has celiac disease, and because of this, maintains a strict gluten-free diet. Ashford and fellow actor Joe Tapper were married at the Devil's Thumb Ranch in Tabernash, Colorado on July 29, 2013. On May 11, 2016, People announced that Ashford and Tapper were pregnant with their first child, a boy. Ashford and Tapper's son, Jack Clark Tapper, was born on September 8, 2016.

Beginning on August 26, 2014, Ashford played the role of Essie Carmichael in the Broadway revival of You Can't Take It with You, directed by Scott Ellis. She appeared alongside Rose Byrne, James Earl Jones, and Elizabeth Ashley. The show closed on February 2, 2015. For this role, she won the 2015 Tony Award for Best Featured Actress in a Play.

Ashford returned to 54 Below for a New Year's Eve edition of her Lost in the Stars show entitled Lost in the Stars: New Year's Magic. The concert also served as an album release party. On November 26, 2015, Lost in the Stars was nominated for a 2015 BroadwayWorld Cabaret Award in the category "Best Show, Celebrity Female". On September 13, 2016, it was announced that Ashford would once again return to 54 Below for an updated version of the New Year's Eve edition of her Lost in the Stars show, Lost in the Stars: New Year's Magic.

On December 13, 2016, it was announced that Ashford would reprise her role as Dot/Marie opposite Gyllenhaal in Sunday in the Park with George in the show's Broadway revival at the Hudson Theater. Ashford was not eligible for a Tony Award for this performance, as the production withdrew itself from any award competition, including the Tony Awards. A statement was released on February 3, 2016 saying: “The producers of Sunday in the Park with George will not be submitting Hudson Theatre’s engagement of this New York City Center production for awards eligibility. With a season so full of tremendous, soon-to-be long-running new musicals and revivals, the producers feel this extremely limited, special run stands most appropriately outside of any awards competition. The production is nevertheless proud to be part of such a landmark Broadway season.”

On November 3, 2017, it was announced that Ashford would be making her New York Philharmonic debut in a televised New Year’s Eve concert celebrating the music of Leonard Bernstein. Joined by Laura Osnes, Aaron Tveit, and Christopher Jackson, New Year’s Eve: Bernstein on Broadway will feature songs from Bernstein's scores for On the Town, West Side Story, Candide, and Wonderful Town. It will be accompanied by Bramwell Tovey, and had aired on PBS on December 31, 2017, at 9:00pm.

On February 15, 2018, it was announced that Ashford would lead a single-camera comedy pilot for ABC entitled Three Rivers. Tristram Shapeero is set to direct.