The former Italy international played the full 90 minutes in the first leg in Turin, but sat out Saturday's win at Benevento with an abdomen injury and reportedly missed training ahead of the trip to Madrid.
Paulo Dybala and Rodrigo Bentancur are also absent due to suspension, although Miralem Pjanic is back.
Federico Bernadeschi is close to a return, but he is not among the travelling party.
Regardless of injuries, coach Massimiliano Allegri is refusing to rule out progression despite Juve needing to overturn a three-goal deficit in Wednesday's clash.
The Bianconeri suffered a 3-0 humbling against the Spanish giants last week and will need to produce one of the greatest performances in their history to book a semi-final berth.
Speaking after his side's 4-2 win over Benevento, Allegri refused to accept that his side's European campaign is over for another year.
"We've got to take every last drop of energy we've got left to go for the Scudetto, Coppa Italia and, of course, the second leg in Madrid," he said.
"We must put in a great performance in Madrid with the world watching and then see what happens."
Allegri also responded to critics of his side and was quick to highlight the strides they have made in European football's premier competition in recent seasons.
"Our European ambitions must be strong, as they always are," he added.
"When I first arrived, there was this terror we wouldn't be able to get out of the group stage. We've since reached two finals in three years.
"If people think Juventus have to win the title and Champions League every year, then that's unrealistic. Barcelona, Real Madrid, Bayern Munich and Manchester City can't manage it, so why should we be expected to?
"That doesn't mean there's a lack of ambition. The Champions League can be transformed by one incident in five minutes, or even by the luck of the draw, but with a league title you have 38 rounds to earn your silverware."