Di Matteo, who won the competition twice as a player with Chelsea, says manager Andre Villas-Boas will field his strongest possible line-up against Portsmouth on Sunday.
He refuses to give up on the Premier League and Champions League trophies, but after Manchester United manager sir Alex Ferguson wrote off the Blues' league title hopes, he admits the onus is on the club to progress in the FA Cup.
"At the moment you look at it and think that maybe the FA Cup is the best chance," he said. "But we are still in the Champions League -- not like some other English teams -- and we are in a better position in the the league than last season.
"We are going to be playing our strongest team, or as strong as we can, looking at the fitness level of the players."
January has brought a flurry of transfer speculation to Stamford Bridge, with Gary Cahill's move to Chelsea nearing a conclusion.
Frank Lampard has reportedly attracted interest from Manchester United, but Di Matteo claims there is little chance of the midfielder leaving Chelsea.
And despite the fact Chelsea will lose Ivory Coast forwards Didier Drogba and Salomon Kalou for the Africa Cup of Nations, Di Matteo expects little activity to take place during the winter transfer window.
"A fee has been agreed for Gary (Cahill) and the negotiations are ongoing with regards to personal terms," he said.
"He has developed into a top central defender in the Premier League. He has made selection for the England national team as well.
"He is at a very good age, so he would add very good qualities to our squad. I wouldn't think there will be much else happening in this market. We are pretty happy with our squad."
On Lampard, Di Matteo added: "I am not surprised other managers like him, but we are very happy Frank is in our team.
"He is a fantastic player and has been for season after season for many many years, for club and country.
"I can't see him leaving this club. He has been here for 10 years and he is part of the successful history of this club.
"I don't know what the future will hold, but at the moment I can't see him going anywhere. He is part of our plans and he will be very important for us."
John Terry is a slight doubt with a knee knock, while Ghanaian international midfielder Michael Essien, who has been out all season with a knee injury, is expected to return to training within the next two weeks.
Portsmouth captain Liam Lawrence claims it is a good time to be facing Chelsea, after their mixed successes over the hectic festive period.
Chelsea lost their last home game 3-1 to Aston Villa, before beating Wolves away in controversial circumstances.
Lawrence said: "They are not doing as well as they would have liked but have still got world-class players with a good manager.
"If we are going to go there at any point in the past few months, I think now would be the time."