Eyebrows were raised when Real boss Ancelotti opted to leave Cristiano Ronaldo out of his starting XI for last Wednesday's last-16 first-leg clash at Vicente Calderon.
Ronaldo was introduced around the hour mark - shortly after Raul Garcia had given Atletico the lead from the penalty spot - but the Ballon d'Or favourite could not inspire a turnaround as Raul Gimenez added a second for the hosts with 14 minutes remaining.
The result represented Real's third consecutive loss to their local rivals, and also condemned them to a second defeat in three days following last weekend's 2-1 Liga reverse at Valencia.
Ancelotti's side - who registered a club-record 22 successive victories in all competitions before losing at Mestalla - got back on track with a routine 3-0 win over Espanyol on Saturday.
And, speaking after that victory, the Italian confirmed that he will not be resting any of his stars on Thursday as Real attempt to keep their bid to retain the Copa alive.
"We have time to prepare for the match well - we'll field our best team becaise we're going to try to turn the tie around and do everything we can to reach the quarter-finals," Ancelotti said.
"Everyone is waiting for this game, but right now we have to be calm.
"We're going to prepare for it, and playing at home is going to help us."
Barring a minor miracle, the victors of the Madrid derby will face Barcelona in the quarter-finals.
Barca put the off-field chaos surrounding the club to one side to hammer Elche 5-0 at Camp Nou in their last-16 first leg.
Neymar, Luis Suarez and the apparently unsettled Lionel Messi all got on the scoresheet.
Valencia have Alvaro Negredo's late penalty to thank for giving them a narrow 2-1 lead to take to Barca's neighbours Espanyol, while Sevilla host Granada having won by the same scoreline at Estadio Nuevo Los Carmenes last week.
Villarreal against Real Sociedad is another tie that hangs in the balance, with Marcelino's side travelling to San Sebastian holding a slender 1-0 advantage.
Elsewhere, Athletic Bilbao entertain Celta Vigo having won 4-2 away last Tuesday, Malaga go to Levante with a 2-1 lead and Getafe take on Almeria with the tie all square at 1-1.
