Mario Gomez, Juan Cuadrado and Josip Ilicic start against Carlos Tevez, Fernando Llorente and Andrea Pirlo in the Europa League.
The second leg of the Round of 16 kicks off at 18.00 GMT and follows on from the 1-1 draw in Turin.
It’s not only an all-Italian European showdown, but also a long-term bitter rivalry between these two clubs.
This is the third meeting in two weeks, as Juve won 1-0 in Serie A and the first leg was last Thursday.
The Bianconeri’s only League defeat of the campaign was at the Stadio Artemio Franchi, when they fell 4-2 in October despite leading 2-0.
Borja Valero is available because his ban only applies to Serie A, while Gomez starts after breaking his post-injury goal drought in the first leg.
Cuadrado was dropped for the game in Turin, but is part of the attack here with Juan Manuel Vargas at left-back, though Mati Fernandez and Giuseppe Rossi are sidelined.
As always with Fiorentina, it can mutate to a 3-5-2 or a 4-3-3 depending on the situation.
Rossi has flown in after his latest check-up and will be in the stands in Florence this evening.
Juventus have to rearrange their defence, as Andrea Barzagli and Angelo Ogbonna are both injured along with Claudio Marchisio.
It means Martin Caceres completes the back three with Leonardo Bonucci and Giorgio Chiellini.
Tevez returns after two games out with a knee tendon problem to partner Llorente, while Pirlo, Arturo Vidal and Paul Pogba form the midfield trio.
There will also be fewer Juve fans than usual at the Stadio Franchi, as there is an ultras boycott in protest at what they deem restrictive measures on this away game.
Fiorentina are unbeaten in nine European games this season, one of only three teams left in the tournament.
Fiorentina: Neto; Tomovic, Gonzalo Rodriguez, Savic, Vargas; Aquilani, Pizarro, Borja Valero; Cuadrado, Gomez, Ilicic
Fiorentina bench: Rosati, Roncaglia, Pasqual, Bakic, Joaquin, Ambrosini, Matri
Juventus: Buffon; Caceres, Bonucci, Chiellini; Isla, Vidal, Pirlo, Pogba, Asamoah; Tevez, Llorente
Juventus bench: Storari, Lichtsteiner, Mattiello, Romagna, Padoin, Vucinic, Osvaldo