The veteran struck a sublime free kick after Gonzalo Rodriguez was sent off to secure a quarter-final spot for the Italian champions
Pirlo has won every major Italian domestic trophy and has experienced much success in European football, but has yet to taste glory in the continent's second tier competition.
But the 34-year-old's hopes of adding that trophy to his list of honours are still intact after he helped Juve overcome Fiorentina for the second time this month, scoring from a free kick again after netting a late winner against Genoa in similar fashion at the weekend.
Juve beat Vincenzo Montella's men 1-0 in Serie A, but it was the hosts who held a potentially crucial away goal going into the game after a 1-1 draw in the first leg in Turin.
Fiorentina had dictated play for much of Thursday's encounter, but the last-16 tie turned midway through the second half when the Florence club saw skipper Gonzalo Rodriguez dismissed after conceding the crucial free kick on the edge of the area.
Pirlo capitalised on the opportunity presented to him, finding the top corner from the set-piece to seal progress to the quarter-finals for Antonio Conte's men 2-1 on aggregate.
Carlos Tevez returned to the Juve starting XI after missing the first leg and the win over Genoa on Sunday, while Mario Gomez was preferred to Alessandro Matri at the head of the Fiorentina attack.
Fiorentina almost made the ideal start inside the first two minutes when Gomez – who scored the equaliser in the first encounter - raced through on goal, but the German could only scuff his shot wide.
The visitors gradually worked their way into the game, although Juve struggled to create any goalscoring opportunities in the first half.
Instead, it was Montella's men who continued to look the more dangerous, David Pizarro's long-range drive forcing Juve goalkeeper Gianluigi Buffon into a fine reflex save just before the half-hour mark.
Tevez and Paul Pogba then drew routine saves from Neto in the Fiorentina goal, before Pirlo curled over with an ambitious free kick from range.
Buffon was the busier of the two keepers for much of the encounter and needed to be on his guard again to keep out a powerful swerving strike from Juan Cuadrado early in the second half.
Yet Juve remained a threat, spurning a glorious opportunity as Arturo Vidal headed narrowly over from Chilean compatriot Mauricio Isla.
However, Juve's hopes were significantly boosted on 69 minutes as Rodriguez was shown a second yellow card for a foul on Fernando Llorente.
And Conte's men immediately took advantage as Pirlo slammed the resulting free kick into the top corner beyond a helpless Neto.
From there Juve were able to take complete control of the contest and were denied a second when Neto produced a fantastic stop from Tevez's close-range header, but the Italian champions held on to maintain their hopes of reaching the final on home turf.