Caretaker Blues boss Di Matteo faces the biggest challenge of his short managerial career this week when he attempts to mastermind a victory over a team billed by some as the greatest of all time.
Chelsea have been written off by some pundits but Di Matteo has spent hours scouring video footage of Barca since both sides won their respective quarter-finals almost two weeks ago.
And the Italian insisted they could be beaten.
"Yes, of course. They do have weaknesses," he said.
"We just need to exploit them."
Doing so has been the problem for almost every team who have faced the Spanish and European champions in recent years but Di Matteo has concentrated his efforts on those who have enjoyed some degree of success.
He added: "You look at the way you can hurt Barcelona and create them difficulties. I've seen many games."
But that has been the limit of Di Matteo's planning thus far, with Chelsea's punishing schedule simply not allowing them to work on specific tactics for Wednesday night's game.
That contrasts sharply with the preparation chronicled by former Manchester United captain Gary Neville ahead of the sides' 2008 semi-final.
Neville revealed United had practised defensive drills until they were blue in the face.
But Di Matteo said: "There has not been time to be able to prepare on the pitch the team against that because we had to prepare for all the other games."
Stopping Lionel Messi, who has scored a simply jaw-dropping 61 goals for the club this season, would appear to be near the top of Di Matteo's agenda.
He said: "It's not just about stopping a single player, because obviously they have so many players that can hurt you.
"It has to be a team tactic, a team strategy, to be able to limit their threat."
Messi is already being dubbed the greatest player of all time.
"You don't need me to tell you how good he is - he's an exceptional player," said Di Matteo, who was reluctant to bracket him above Pele, Diego Maradona or even Cristiano Ronaldo.
"It's difficult to compare, because of different eras they played in.
"I watched Pele on TV. I watched Maradona personally and Messi now.
"You can put Ronaldo in that category as well.
"We're just talking about great players in the history of football."
Di Matteo also refused to get carried away with talk of Barca being the greatest team of all time.
He said: "Certainly, one of the best teams in the world. They show it by all the success they've had.
"Over the years, certainly in the last five or six years, they've been at the top.
"Bayern Munich is probably up there, Real Madrid is probably up there, AC Milan is probably up there.
"I think they might have a little edge but I think we're talking about fantastic football teams here."
Chelsea's best chance of success against Barca would appear to be exploiting the superior physicality that so nearly saw them beat the Spanish giants in their last, highly controversial, meeting three years ago.
The Blues are likely to spend long periods without the ball but Di Matteo had no fears about how supporters might react to that.
"The fans will be behind us, no doubt about it," he said.
"The fans have always been great at Stamford Bridge, they've always been very supportive to the team and I've absolutely no concerns about the fact that the fans will be cheering the team on."