Without the insurance of the combative De Rossi, the Italy coach has brought Claudio Marchisio and Angelo Palombo into midfield.
"I thought seriously about playing two strikers with Antonio Cassano behind. Then without De Rossi I had to alter my idea. He doesn't have a clone," Prandelli told a news conference on Monday, saying other changes followed watching DVDs of Serbia.
Striker Giampaolo Pazzini will replace Marco Borriello, who lacked conviction in front of goal in Friday's 0-0 draw in Northern Ireland which kept Italy top of Group C.
Right back Gianluca Zambrotta, who thought his Azzurri career was over after the World Cup, will pick up 98th cap in place of Mattia Cassani and winger Simone Pepe has been dropped.
Stefano Mauri, who impressed in his all-action display on Friday in his first Italy appearance since 2007, is likely to move into Pepe's role out wide with forward Cassano teaming up with Pazzini on their Sampdoria home ground.
TRICKY MATCH
Centre-back Leonardo Bonucci has a slight foot problem but is expected to play.
Prandelli, who took over from Marcello Lippi after the then holders crashed out early in the World Cup, has been revealing his team a day before games but his Serbian counterpart said the game was too important to give away secrets.
Vladimir Petrovic, whose side were embarrassed 3-1 at home by Estonia on Friday in his first game in charge, said: "I can't tell you much about the formation, it's a fundamental game. I can say that Neven Subotic will be on the field, he is a valid alternative to the suspended Nemanja Vidic."
Serbia, who have four points from three games in fourth, expect a tricky match in Genoa.
"A draw would be a good result," the coach added. "It will be a difficult game for both sides, both of us played badly in the last match."
Italy team: Emiliano Viviano; Gianluca Zambrotta, Leonardo Bonucci, Giorgio Chiellini, Domenico Criscito; Angelo Palombo, Andrea Pirlo, Claudio Marchisio; Stefano Mauri, Antonio Cassano, Giampaolo Pazzini
