Born and raised in Trenton, Michigan, Rajskub later moved to Los Angeles where she became one of the original cast members of Mr. Show, and also had roles on The Larry Sanders Show, Shasta McNasty, and Veronica's Closet.

In her time on Mr. Show, she was the real-life girlfriend of David Cross, leaving the show after the second season when they broke up.

She has since appeared in more dramatic roles, most notably her role as CTU tech analyst Chloe O'Brian on 24, which she joined in 2003 at the start of the show's third season.

Her character was a hit with viewers and critics and was one of the few cast members to return in the show's fourth season. After being a regular guest star for two seasons, Rajskub became a main cast member in the show's fifth season. For the sixth season, she replaces Kim Raver as lead female, with top billing second only to Kiefer Sutherland.

She also appeared in Kelsey Grammer's The Sketch Show on Fox Television and numerous films including Serial Slayer (aka Claustrophobia), Mysterious Skin, Legally Blonde 2, Sweet Home Alabama, Dude, Where's My Car?, Man on the Moon, Punch-Drunk Love, The Anniversary Party, Firewall, Little Miss Sunshine, Weezer's music video for their song The Good Life, as well as portraying a blind girl in the film Road Trip.

She is also a skilled guitar player, and was part of a comic duo (with Karen Kilgariff) called Girls Guitar Club. In 2006, she made a cameo appearance in an episode of Gilmore Girls where she played a troubadour looking for her big break. (Rajskub had previously appeared on Gilmore Girls as the female lead in A Film by Kirk, a short film made by the character Kirk Gleason).