The club lodged an official complaint with the Football Association before the match alleging Mark Clattenburg, the referee from Chelsea's stormy 3-2 league loss to United on Sunday, used inappropriate language towards the Nigerian.
Players' union chief Gordon Taylor has said a comment made by the referee during Sunday's game was of a racist nature. The referee has not commented publicly.
Mikel had a strong match in the centre of Chelsea's midfield on Wednesday, before being withdrawn in the second half as he had picked up a yellow card.
Di Matteo would not answer questions from reporters about the Clattenburg issue, but said he was confident Mikel was mentally right to play.
"I spoke to my players in the last couple of games and mentally they were in the right frame of mind to play, so I had no concerns picking the team I wanted to," the Italian told a news conference.
"He [Mikel] is fully focused on playing football."
Chelsea have drawn traditional foes Leeds United in the quarter-finals, with the match taking on extra intrigue following Leeds manager Neil Warnock's comments that he was "disgusted" by Chelsea's actions against Clattenburg.
Di Matteo would not respond to questions on that issue either, but said he was expecting a tough encounter.
"It's always difficult to go and play away from home, Leeds and Chelsea, they have a little bit of history there, it will be an interesting game for us to go and play at Leeds," he said.
