According to NFL Network's Ian Rapoport, the Colts elevated safety Rolan Milligan from the practice squad to the 53-man roster in place of Mitchell.
Indianapolis will face the top-seeded Kansas City Chiefs on the road Saturday for a chance to play in the AFC Championship Game.
The 31-year-old Mitchell is in the midst of his first season with the Colts after he spent the previous four campaigns with the Pittsburgh Steelers. During the regular season, Mitchell appeared in eight games and made four starts, finishing with 28 tackles, two forced fumbles and one interception.
Mitchell's veteran presence may be missed Saturday since he has participated in nine playoff games during his 10-year career with the Oakland Raiders, Carolina Panthers, Steelers and Colts.
The 24-year-old Milligan has never appeared in an NFL regular-season contest and spent time in the Dallas Cowboys, Detroit Lions and Colts organizations.
If the Toledo product is active Saturday, it will mark his first NFL game outside the preseason.
Malik Hooker and Clayton Geathers will be the starting safeties against Kansas City if they can play, but they are both nursing injuries. Neither Hooker nor Geathers practiced Tuesday because of foot and knee ailments, respectively.
Because of those setbacks, unheralded players such as J.J. Wilcox, George Odum and Corey Moore could see significant action.
Whoever is present in the Indianapolis secondary will be tested in a big way against Chiefs quarterback Patrick Mahomes, who threw for 5,097 yards and 50 touchdowns during the regular season.