The Portugal captain and Ballon d'Or winner has missed Real Madrid's last three matches since limping off near the end of the Champions League quarter-final, first leg against Borussia Dortmund on April 2.
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 tomorrow."
The latest edition of Spain's 'Clasico' between arch-rivals Real Madrid and Barcelona FC 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.
"I don't think there will be any problem motivating the players tomorrow, they are all very motivated and very excited. I am seeing a lot of desire to play this final."
Ancelotti dismissed a suggestion Real Madrid could have avoided Ronaldo's injury if they had given him more rest.
"Cristiano did not rest because there was no need," he told reporters.
"There was no situation we saw that suggested he was tired and if he had been tired he would not have played.
"He has a problem that many players have at this stage of the season.
"We protect Cristiano in the same way we try to protect all the players as much as possible.
"It could be a risk playing him and we are not going to take that risk as there are very important games still to come."
Ancelotti's Barcelona FC counterpart Gerardo Martino also has injury worries and is waiting on the fitness of three of his recognised centre backs.
Captain Carles Puyol, who has barely featured this term due to a series of injuries, Gerard Pique and Marc Bartra were all included in the squad for the trip to Valencia and Martino said a decision would be taken on Wednesday whether they could play.
Barcelona FC's Spain playmaker Andres Iniesta said he expected Real Madrid to be just as much of a threat without Ronaldo.
"The team doesn't change whether he is playing or not," Iniesta told a news conference.
"We know how good they are and we know their potential. If Cristiano doesn't play somebody else at the same level will."
Barcelona FC, the record winners with 26 Cups, are looking for a morale-boosting victory after crashing out of the Champions League to Atletico Madrid on Wednesday.
The followed that up with a shock La Liga reverse at Granada on Saturday that left them four points behind leaders Atletico and one adrift of Real Madrid with five games left.
FACTBOX: Real Madrid v Barcelona FC - the 'Clasico' in numbers
MADRID, April 15 (Reuters) - Real Madrid and Barcelona FC meet in Wednesday's King's Cup final in Valencia (1930 GMT) in the latest edition of the 'Clasico' between the world's two richest clubs by income. Here are some numbers behind one of soccer's most glamorous fixtures:
0 - The number of goals Real Madrid have conceded in eight matches on the way to the Cup final.
2 - The number of 'Clasico' games Real Madrid coach Carlo Ancelotti and his Barcelona FC counterpart Gerardo Martino have each presided over. Barcelona FC beat Real Madrid 2-1 at the Nou Camp at the end of October and won 4-3 at the Bernabeu last month, both in La Liga.
2 - The number of hat-tricks Barcelona FC's Argentina forward Lionel Messi has scored in 'Clasicos'. Messi netted a treble in his first 'Clasico' appearance at the age of 19, a 3-3 La Liga draw at the Nou Camp in 2007, and grabbed another in last month's league clash.
18 - The number of cards the most booked player, former Real Madrid and Spain defender Fernando Hierro, collected in official 'Clasicos' (17 yellow and one red). Current Real Madrid centre back Sergio Ramos has 14 yellow and two red.
21 - The number of goals Messi has scored in 'Clasicos'. The four-times World Player of the Year is the record marksman in the fixture ahead of Real Madrid great Alfredo Di Stefano on 18. Current World Player Cristiano Ronaldo has 13 for Real Madrid, including the extra-time winner in the 2011 final. Messi has not scored in five Cup matches against Real Madrid.
22 - The number of times Real Madrid and Barcelona FC have played each other in the Spanish Cup. Barcelona FC have 10 wins to Real Madrid's six.
26 - The number of King's Cups won by record winners Barcelona FC, three more than Athletic Bilbao in second with 23. Real Madrid are third with 18.
44 - The record number of 'Clasico' appearances made by Real Madrid's Paco Gento. Among active players, Barcelona FC playmaker Xavi has the most with 39, ahead of Real Madrid goalkeeper Iker Casillas on 34 and Barcelona FC defender Carles Puyol on 32.
90 - Real Madrid have beaten Barcelona FC 90 times in official matches, with 89 wins for Barcelona FC and 48 draws.
227 - The number of official 'Clasicos' played since the pair's first encounter in the last four of the Spanish Cup on May 13, 1902, which Barcelona FC won 3-1.
383 - The number of goals Real Madrid have scored in official 'Clasicos', 13 more than Barcelona FC.