The Portugal captain and Ballon d'Or winner has missed Real's last three matches since limping off near the end of the Champions League quarter-final, first leg against Borussia Dortmund on April 2. (Agencies)
Ancelotti said on Friday there was a still a chance Ronaldo might recover for the clash at the Mestalla stadium in Valencia but confirmed at a news conference the player was unavailable.
"Cristiano Ronaldo is out, we have a lot of players sidelined," said Ancelotti, who will also be without longer-term absentees Jese, Sami Khedira, Alvaro Arbeloa and Marcelo.
"But we have a lot of confidence in this team," added the Italian, who declined to answer a question about who would start the Cup final in Ronaldo's absence.
"It has always reacted well to injuries and I think we can also do things very well on Wednesday."
The latest edition of Spain's 'Clasico' between arch-rivals Real and Barca is their 228th meeting and a repeat of the 2011 Cup final, also held at the Mestalla, when Ronaldo's extra-time header secured a 1-0 win.
The Portuguese is top scorer in La Liga and the Champions League this season and Ancelotti will be desperate to have him back for the semi-finals of Europe's elite club competition later this month when they play holders Bayern Munich over two legs.
"We are losing a player with incredible quality. But it could be that Cristiano Ronaldo's absence will motivate the rest of the team even more," Ancelotti said.
"The matches we have played without him have gone very well, working harder, running more and working together more. Working as a unit is the of course the objective and that is what we have been training for,” he said.
"I don't think there will be any problem motivating the players on Wednesday, they are all very motivated and very excited. I am seeing a lot of desire to play this final,” he added.
The Portugal captain and Ballon d'Or winner has missed Real's last three matches since limping off near the end of the Champions League quarter-final, first leg against Borussia Dortmund on April 2.