Versatile Italy international Florenzi, who typically plays on the right side of defence for Roma, has started 20 Serie A games out of 25 for Eusebio Di Francesco's side this season.
Illness could keep Florenzi out of the game and, with right-back Rick Karsdorp out long-term, Bruno Peres is an option to fill in, the Brazilian having been sent off against Atletico Madrid in the group stage.
Although Florenzi has travelled to Ukraine with the rest of the Roma squad, Di Francesco confirmed the 26 year-old is unable to train.
"Alessandro has stayed in the hotel because he's not well," the Roma manager said. "Hopefully, he'll be available for selection but we'll have to see how he is tomorrow morning.
"If the game was today he wouldn't play and he won't train later either."
A change of formation and the form of Turkish forward Cengiz Under - the 20-year-old scoring four goals in three games - has helped Roma to three successive Serie A wins ahead of the Shakhtar tie.
But Di Francesco, while highly impressed with the youngster's performances, would not give any indication whether Under has earned a Champions League debut.
"Cengiz has been sensational lately - he's really come on in leaps and bounds," Di Francesco added. "He's been key for us but I prefer to focus on the team rather than individuals.
"It's Roma who are going out there to play tomorrow, not Cengiz. We're delighted to have him here and we'll try to exploit his talent as best we can.
"I'm not going to tell you who will play tomorrow. I want to keep all the lads on their toes and I want everyone to feel important and involved.
"We've significantly upped the intensity of our training sessions. It adds a small element of risk but the idea is to fill up the tank for the business end of the campaign and I hope to see the benefits on the pitch.
"We must play with grit, determination and belief – they're all things that can make a difference out there."
After knocking out Atletico Madrid in the group stage, many will expect Roma to come through their Shakhtar tie and reach the quarter-finals for the first time since 2007-08, but Di Francesco believes his side face a tough test.
"We've earned the right to be in the knockout stages and we want to make the most out of it now but we're up against a very talented team who are used to playing at this level," Di Francesco said.
"We have a 50 per cent chance of going through and hopefully we'll be able to take a good result back to Rome for the return. For now, though, we're focused solely on this game.
"We have to try and maintain a high tempo throughout the game. There will be times when we're in control of proceedings but we're up against a side who are very good in possession. We must stay strong as a unit.
"We've been looking a lot better recently and I hope to see further improvement tomorrow."