A year and a half after the couple announced their split, the actor has filed divorce papers at Los Angeles Superior Court.
Will has asked the court to award Amy and him joint physical and legal custody of their two sons Archibald and Abel.
The couple announced they were separating in September 2012 after nine years of marriage.
"All people have been doing since the dawn of time is trying to figure out how to live this life and be happy," Will said after their split. "I'm no different. I'm 43, and I've found my happiness — which is my kids."
Will and Amy, 42, have collaborated together on a number of films, including Blades of Glory and Horton Hears A Who!.
Amy and Will in 2004
The Arrested Development actor has been married once before; he was divorced from Penelope Ann Miller after one year of marriage in 1995.
Amy and Will tied the knot on 29 August, 2003. They welcomed their first son Archie in October 2008, and Abel followed in August 2010. In September 2012, the couple announced their separation.
Following their split, Will was briefly linked to Billy Joel's ex-wife Katie Lee. Amy, meanwhile, has been dating actor Nick Kroll since early last year.