Ronaldo was a major doubt for the game at the Mestalla and Ancelotti explained at his pre-match press conference that the Portugal international will instead focus on being fit for the first leg of the Champions League semi-final against Bayern Munich next week.
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Ancelotti said: "Cristiano is not availabe because we don't want to take risks and we have other very important games this season. He will do individual work (on Wednesday) and then travel to watch the game."
The Italian has been criticised for not resting Ronaldo earlier in the campaign to prevent him from getting injured at the crucial stage of the season, but insisted: "I don't think that would have changed anything. With him or without him in the team, we have the same idea of how we want to play.
"Cristiano didn't rest because he didn't need to rest. There was no situation where we saw him being too tired. At this stage of the season, many players have this problem and we have taken as much care with him as we have with all the other players."
And Ancelotti believes that, even without their talisman, Real still have every chance of beating Barcelona.
"We have many players out, but we have a lot of confidence in this team and we have always responded well when players have been missing, and I am sure we will do the same (on Wednesday)," he said.
Ancelotti has lost both of his battles with the Catalans in his first term in charge of Madrid, but said he was not motivated by a sense of revenge.
"When you play a final, you are obliged not to lose," said the 54-year-old, who will welcome captain Iker Casillas back into his starting line-up. "There is nothing more to say than that. We know anything can happen in a final and we hope to be the better team."
Gerardo Martino's men are preparing to face their arch rivals after a week in which they were eliminated from the Champions League by Atletico Madrid and had their Primera Division title aspirations dealt a devastating blow with defeat at Granada.
The two results were the latest chapters in a tumultuous campaign, which has witnessed the resignation of president Sandro Rosell, a FIFA-imposed transfer ban for breaching its rules on the international transfer of minors, plus a host of injuries to key players.
However, speaking at the pre-match press conference, midfielder Andres Iniesta said: "We always try to isolate ourselves from what has happened, even though it is very difficult.
"If you analyse the season from day one, we have had to endure incredible things and deal with big setbacks that have been very hard to take - it's not been easy.
"But we have to overcome it all, because that's life - it's about survival and you cannot stop to think. We are ready to play this final."
Iniesta rejected suggestions that Real headed into the final as favourites, given Barcelona's recent dip in form.
"My personal feeling is that Barcelona are not down in the dumps," he said.
"We have had a week with two tough moments, but we have to put it behind us, even though it's difficult.
"This game is a final - there's a trophy at stake. It's another competition and a chance to make the fans happy. We cannot allow ourselves to think about anything else other than this final and winning this trophy.
"We are convinced we can do that, but if we want to we have to be at our best against a powerful rival. The team is capable of playing well and winning and that's what we're going to try and do."
Martino was forced to play midfielder Sergio Busquets in defence in the defeat at Granada and may have to do the same against Real Madrid, with Marc Bartra still not 100 per cent following a hamstring problem.
Bartra was able to participate in the final training session before the final - unlike veteran defender Carles Puyol, who has a knee injury, and Gerard Pique, who suffered a hairline fracture to his pelvis two weeks ago.
However, all three defenders will travel to Valencia - despite not having the all-clear from doctors - with Martino insisting each had a chance of recovering in time.
The Argentinian also stressed that winning the silverware was enough motivation for his team, regardless of the recent defeats.
"We want to win because it's the Copa del Rey, because it's a trophy and because the only reason you play in a final is to win it - it has nothing to do with revenge," he said.
"We will have to find responses during the game but we don't need to respond to what has happened in recent weeks. This is a different game. What has happened has happened, and (Wednesday's) game is a final and we have to win it."
Martino also played down the impact of newspaper stories implying he would lose his job in the summer, with Borussia Dortmund coach Jurgen Klopp being touted as a possible successor.
"It doesn't bother me and I understand that it is logical. I have learned to deal with stories like that all season. People said I wouldn't last until Christmas, but here we are at Easter, and I'm still here."