Diego Simeone's men are wobbling for the first time this season, after suffering a shock Liga loss against Almeria just days after being routed by their city rivals.
Atletico Madrid has little hope of reaching the Copa del Rey final but will want to lay down a marker against Real Madrid on Tuesday.
The Spanish capital rivals go into the semifinal tie's second leg at Vicente Calderon with los Blancos 3-0 up on aggregate, leaving Atleti facing a mountain to climb if it is to retain the crown they claimed last term.
However, Diego Simeone's side will have plenty to play for, if they use the clash as a dress rehearsal for the sides' Liga derby on March 1. That game takes on huge significance given the incredibly tight nature of the Primera Division title race.
The Rojiblancos led the league outright for the first time since 1996, after beating Real Sociedad last weekend, but were knocked off that perch on Saturday as they slumped to a 2-0 defeat at Almeria, while Madrid moved above them courtesy of a 4-2 win over Villarreal.
Atleti captain Gabi - who ended the Almeria defeat in goal after Dani Aranzubia was sent off - has challenged his colleagues to snap their bad run immediately.
"The team is gutted," the midfielder said. "But you have to raise your head, work harder if possible to remove this slump soon after two matches. Let's see the power this team has (to) overcome it.
"Now we have to be united more than ever. We have been in a privileged position, and we are lucky to still be there and we will fight because this little bad run is costing us points."
Atletico won at the Bernabeu earlier this season, its first league triumph over its neighbors in the 21st century, as well as last season's Copa final - but the sides' head-to-head record is massively stacked in Madrid's favor.
Carlo Ancelotti's men will take further confidence from the fact that star man Cristiano Ronaldo is likely to return to their XI as his current Liga suspension does not affect his Copa availability.
The Portuguese attacker could be reunited in attack with Gareth Bale - who returned from a calf injury with a goal and assist against Villarreal.
Atletico, meanwhile, will be without Tiago, who suffered a sprained knee against Almeria, with David Villa (hamstring) and Filipe Luis (groin) also sidelined.
One bright spot for Simeone will be the return of Koke, available again after suspension forced him out of the weekend's defeat.