Juve went down 2-1 at home to the Viola in the first leg of the Coppa Italia semi-final at the Juventus Stadium in their first defeat since October last year.
But Padoin is adamant his side must show a positive reaction when they face Sassuolo on Monday
"We need to bounce back from this defeat and I’m sure we’ll do so at the first time of asking on Monday,” Padoin told the club website.
"They were the first side to bring an end to our run of six consecutive victories at the start of the season, but winning is the most important thing on Monday.
“[The Fiorentina] result may have been a setback, but the tie’s not over. In a month’s time we’ll travel to Florence looking to overturn the scoreline.”
The Bianconeri have averaged 2.7 goals and grabbed two wins and a draw in their last three games against Sassuolo, who come into the game on the back of three consecutive Serie A defeats.
Nine points clear of Roma at the top of Serie A, the Old Lady are on course for their fourth consecutive league title, but are desperate to improve their Champions League performance after failing to escape their group last season.
They take a narrow 2-1 first-leg home victory to Borussia Dortmund later this month, a game that Padoin insists his side must try and win.
"We have a slight advantage because two of three outcomes would see us go through,” he said.
"Having said that, I think it would be wrong to travel to Dortmund and aim for a draw, because you risk losing by playing with that mentality.”