She began her musical career as the lead singer for the Latin freestyle group Pajama Party, placing three songs on the Hot 100 in 1989 and 1990. As a solo artist her biggest success is on the Hot Dance Music/Club Play chart, where in 1996 she hit #1 with the song "I Found It." She returned to the top of the dance/club play charts in 2003 with a dance version of Elton John's "Rocketman". In 2001 she recorded her debut full-length rock album of original songs ("Souvenirs") but it fell through the cracks when her record company Mercury merged.
Rubin-Vega performed in the Broadway musical Rent, starring as Mimi Marquez, heroin-addicted nineteen-year-old with HIV who works at the Cat Scratch Club as an exotic dancer. One of her castmates was Wilson Jermaine Heredia, with whom she also starred in the 1999 film Flawless. She left the cast on April 5, 1997, and was replaced by Marcy Harriell. Rubin-Vega did not participate in the film adaptation of Rent, as she was pregnant at the time of the movie's casting and filming. The role was subsequently given to Rosario Dawson.
Rubin-Vega has two Tony Award nominations to her credit: one for her role in Rent as Best Actress in a Musical,and the other for her performance as Conchita in Anna in the Tropics (2003), as Best Performance by a Featured Actress in a Play. She won the Theatre World Award in 1996 for Rent. She was also awarded the Blockbuster Award for Best Supporting Actress in a Suspense Thriller for her role in the film Wild Things with Kevin Bacon and Neve Campbell.
She appeared in the 2000 Broadway production of The Rocky Horror Show in the role of Magenta.
Rubin-Vega also released her second full-length album of original songs titled Redemption Songs released on October 2006 on Sh-K Boom Records.Her rock band DRV is currently performing live shows in New York. She starred with Phylicia Rashad in a musical version of Federico García Lorca's The House of Bernarda Alba at Lincoln Center in March 2006. She played the role of Fantine in the 2006 Broadway revival of the popular musical Les Misérables beginning November 9.On March 2, 2007, she was replaced by Filipino Tony Award winning actress Lea Salonga.
In February 2007, Daphne Rubin-Vega performed alongside Philip Seymour Hoffman in the play Jack Goes Boating off-Broadway at The Public Theater and also appeared in the film version. Rubin-Vega appeared in a cameo role in Sex and the City: The Movie, which premiered in May 2008.
In November 2010, she received the Independent Spirit Awards nomination for 2011, for her role in Jack Goes Boating. The award ceremony was held in Santa Monica, California in February 2011.
She is currently starring Off-Broadway as Yvette in Tommy Nohilly's world premier of Blood From A Stone at The New Group's Acorn Theater until February 19, 2011. She is scheduled to appear in the Off-Broadway cast of Love, Loss, and What I Wore from March 23 to April 24, 2011.
In 2011, the feature film Union Square, co-written and directed by the Sundance Film Festival's Grand Jury Award Winner, Nancy Savoca, is being premiered at the Toronto Film Festival. In it, Daphne co-stars with Mira Sorvino, Patti Lupone, Tammy Blanchard, Mike Doyle, and Michael Rispoli.
In spring 2012, Rubin-Vega will return to Broadway in a new revival of Tennessee Williams' A Streetcar Named Desire, in which she will play the role of Stella Kowalski opposite Blair Underwood as Stanley. This revival will be directed by Emily Mann and features a mostly all-African-American cast.