This week's clash will conclude a hat-trick of meetings between the sides in less than a fortnight, with Juventus taking the spoils in the opening Serie A encounter before a 1-1 draw in Turin last Thursday put Fiorentina in the driving seat in the continental tie.
Arturo Vidal handed Juve the lead in the third minute of the first-leg clash, but Mario Gomez found the net 11 minutes from time to give Fiorentina the away-goal advantage.
However, promisingly for coach Antonio Conte, Juve have only failed to progress once when taking a 1-1 scoreline into the second leg of a European tie.
The only time they did fail to make it through was in 1972, when a 2-1 second-leg defeat at Wolves - now competing in England's third tier - sent them out of the UEFA Cup, but they have never beaten Italian opposition away from home in European competition.
And the Turin outfit will be especially keen to make it through this season, with Juventus Stadium set to host the competition's final.
Conte's men sit top of Serie A and are on an 11-match unbeaten run in all competitions, but lost 4-2 when they played at Stadio Artemio Franchi in the league earlier this season - a result that remains Juve's only top-flight defeat of the campaign.
Fiorentina, meanwhile, brought an end to a five-match winless run with a 3-1 home victory against Chievo on Sunday, and goalkeeper Neto insists his side do not fear their familiar opponents.
"When there are games of this type in Florence the atmosphere is incredible, it's always like that," he told Sky Sport 24. "We have no fear, there are two teams of a high level going up against each other.
"They have a lot of champions, but so do we.
"(Juve goalkeeper Gianluigi) Buffon is of the highest calibre, it is an honour to play against him."
Andrea Barzagli (calf) is a doubt for Juve after pulling out of Sunday's 1-0 win at Genoa, while fellow defender Federico Peluso (thigh) is also likely to miss out. In more positive news, striker Carlos Tevez has returned to training this week and could be in line for a start.
Fiorentina striker Giuseppe Rossi (knee) is expected to be in attendance in a spectating capacity on Thursday, having returned to Italy from the United States as he continues his rehabilitation from ligament damage.