Ronaldo, who has scored 31 goals in the Primera Division this season and hit a hat-trick in the corresponding fixture 12 months ago, sustained a minor injury to his left thigh during the warm-up before Madrid's 4-1 defeat to Borussia Dortmund in the first leg of their Champions League semi-final tie during the week.
He has undergone physiotherapy with the aim of being ready to face the Germans next Tuesday and will be rested against Los Rojiblancos.
Full-back Alvaro Arbeloa has not recovered from back pain and is expected to miss the match too, adding to Madrid's problems at full-back, with Marcelo and Michael Essien also out, meaning Sergio Ramos is likely to continue to play at right-back.
Atletico are currently three points behind Madrid in the Primera Division table and victory would take them level on points with Los Blancos with five games of the season remaining.
Madrid's injury problems, plus the fact coach Jose Mourinho is likely to rest further players ahead of the second leg against Dortmund, means that Diego Simeone's side have an excellent chance of beating their neighbours for the first time since 1999.
Atleti captain Gabi told a press conference that the players were fired up to finally get one over their rivals.
"We have 14 years of desperation to win this game," he said.
"Real Madrid are a special rival because of what it means to play against them and the excitement around the fixture.
"It has been a long time since we arrived at a derby so evenly matched and with the opportunity to go level on points with them, and it's been a very long time since we beat them."
The midfielder emphasised the good form his side are in and believes they have what it takes to end their barren run against Madrid.
"It's clear we are in a very good moment, we have just beaten Sevilla away from home and we are focused on the task ahead. On Saturday we have a great opportunity.
"We have spent a long time talking about the best way to win a derby and now we have to go and do it."
Gabi is hopeful Mourinho will make major changes but warned the visitors will still pose a major threat.
"We expect that Madrid will come here to win the game, regardless of which players play," he said.
"No-one expected that result (against Dortmund), not them, nor the people who watched it from the outside, but they know how to get over this.
"It is obvious that at the end of the season those special players like Cristiano or [Mesut] Ozil are the ones that win you most points. If Mourinho doesn't think they should play then it is better for us."
During the week Atletico forward Diego Costa was cleared to play after the yellow card he received against Sevilla last week, which meant he would have missed the game with Madrid, was rescinded.
Simeone now has a full squad to choose from, with the exception of Arda Turan, who is still recovering from an ankle injury.