The interim Blues boss has to decide whether to stick with Didier Drogba for Wednesday night's first leg at Stamford Bridge or recall Fernando Torres for the crunch clash.
Based on current form and previous meetings with Barca, Drogba is the obvious choice.
But that would also be the case for Saturday's crucial Barclays Premier League game at Arsenal and the following Tuesday's return leg against Barcelona.
Chelsea's punishing schedule has so far seen Di Matteo give almost equal game time to Drogba and Torres and, after the former started in Sunday's FA Cup semi-final thrashing of Tottenham, it would appear something has to give in the coming week.
Whether that is on Wednesday or on Saturday or next Tuesday remains to be seen, but Drogba, who has long been used to being top dog at Stamford Bridge, has accepted that rotation is a fact of life for the rest of this season.
"It's new for me, but there's nothing bad about it," he said.
"The most important thing is for the team to win games and to go forward.
"Of course, I would like to play more, but I think the other players would like to play more as well, so I can't complain."
Drogba is not about to volunteer for a rest, however.
He added: "All these games now are very important for us, the ones in the league, the FA Cup final, the ones in the Champions League.
"That's why you sign for Chelsea, because you want to play all these games and this is what is really exciting for me."
Midfielder Frank Lampard's playing time under Di Matteo has seen him start two games in a row, miss the next, and then start the next two in a row.
If that sequence continues, he will be left out on Wednesday.
"I'm not going to be asked to be rested, no, no, no!" he said.
"I think we live right these days and the idea of another two or three days between the game and then playing Barcelona, if you can't get your body ready for that and your mind ready for that then I don't know how you can do anything.
"The manager will pick that team on its merits on Wednesday.
"We're fit and fresh and ready to go."
The 33-year-old fell out with the sacked Andre Villas-Boas over his lack of game time, but has been more willing to sit out matches under Di Matteo.
Lampard has previously suggested he would have bitten his tongue under Villas-Boas had Chelsea not been losing and Drogba said: "It's easier when you win games. The spirit is better when you win games.
"That's what we're doing at the moment and, so, fingers crossed - I hope we can continue."