Messi missed Argentina's two friendlies last month with a hamstring injury and was only a substitute in Barca's 2-2 draw against Sevilla on March 31, but he was back in the starting line-up for the 4-1 victory over Roma at Camp Nou last Wednesday.
The 30-year-old was not among the goals in the first leg but hit a hat-trick in a 3-1 win against Leganes on Saturday, Ernesto Valverde opting against resting the superstar.
Di Francesco felt Messi's latest display proves how effective Roma were in keeping him quiet and wants a repeat at the Stadio Olimpico on Tuesday.
"Everyone was saying he wasn't fit and suddenly he scored a hat-trick," the Roma coach told a pre-match news conference.
"This means we managed to limit his movement between the lines. We have to reduce the space around him as much as possible, wherever he pops up on the pitch.
"Barcelona's strength comes from their mentality, which starts from the youth academy and has been built up over the years. They manage games superbly, using the ball well and making few mistakes.
"We forced them into errors in the first leg, but we didn't take advantage of it. On the other hand, we paid dearly for our mistakes. We want to control the game but not concede."
Radja Nainggolan missed the first leg with a thigh injury but returned for the 2-0 loss to Fiorentina on Saturday.
Despite having a crunch Serie A clash against arch-rivals and fellow top-four side Lazio scheduled for Sunday, Di Francesco has no plans to rest any key individuals.
"I think we should choose our strongest line-up, it's important and we must believe," said the coach.
"Let's perform with great heart and passion. We have to honour and respect our club colours, so we'll give it our all and we deserve the chance to achieve something huge.
"Why not believe until the end and hope we can achieve something unprecedented? We're trailing by three goals, so it's a really tough ask, but we have the duty to try. So, our best team will start and then we'll think about the derby."
(goal.com)