The left-back sat out of training on Monday, and was expected to be a doubt for the clash after suffering a problem with his hamstring.
Di Matteo has now confirmed that the 31-year-old will miss the tie, but insisted that he has every faith in Ryan Bertrand's ability to deputise.
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"Ryan Bertrand has always performed well for us when he's played and I have full confidence he will play a great game."
However, the Italian went on to warn his players that every one of them must be at their best if the Blues are to succeed in beating the Ukrainian champions, and suggested that his side's winning mentality could aid their task.
"We need to be at our best, every player has to perform to the best of their ability. The early goal in Donetsk helped them," he continued.
"The team has a winning mentality and experience of performing in big games like this, which helps. We have very good players."