Acker made her major TV debut on Angel starring as Winifred Burkle from the 2nd to the 5th season, as well as portraying Illyria for the 5th and final season. She joined the cast of Alias in 2005 for the final season as a villain. In the same year, Acker provided her voice for the character Huntress on the animated series Justice League Unlimited.
Acker guest-starred on the How I Met Your Mother. She portrayed Dr. Claire Saunders/Whiskey on the show Dollhouse. She guest starred in 10 of the 13 episodes of the first season as well as 3 of the second season.
In 2010, Acker became a series regular on ABC's drama Happy Town portraying Rachel Conroy. She also starred in the season one finale of Human Target as well as CBS's Person of Interest.
Acker appeared in horror movie Cabin in the Woods in 2012. She also had roles in shows such as Grimm, Warehouse 13, Supernatural, and Once Upon a Time. She appeared as Beatrice in the film Much Ado About Nothing.
Acker appeared in the sitcom Husbands's third season. In 2014, Acker was cast as Audrey in an episode of Agents of S.H.I.E.L.D. Between 2012 and 2016, Acker portrayed Samantha "Root" Groves on the CBS drama Person of Interest.
In 2017, Acker was cast as Caitlin Strucker in the FOX television series The Gifted, until its cancellation in 2019.
Initially as the scientist Winifred "Fred" Burkle, Acker portrayed the Old One Illyria in the final 8 episodes of the series. She first guest starred in 3 episodes of Angel then joined the main cast for the 66 following episodes:
- "Over the Rainbow"
- "Through the Looking Glass"
- "There's No Place Like Plrtz Glrb"
- Angel season 3
- Angel season 4
- Angel season 5
- Amy Acker on Wikipedia