Carlo Ancelotti's men will be looking for Copa del Rey revenge, although their local rivals' Liga form suggests they will be in for a stern semifinal test.
The two sides, which met in last season's Copa del Rey final, face each other in the first leg of the competition's semifinals, with Diego Simeone's side having won the last two meetings.
If the Rojiblancos can repeat the feat at the Santiago Bernabeu it would be the first time they have ever won three consecutive matches against Real, and would boost the holders' chance of retaining the trophy.
Having ended Atletico's 17-year wait for the trophy, Simeone has continued to revolutionize the team, guiding it to the top of La Liga's table for the first time since 1996.
The visitors, who beat Real Sociedad last time out, are now unbeaten in 23 matches in all competitions, underlining the size of the task facing Carlo Ancelotti's men.
But, although Simeone's side has won the last two clashes, it is the Blancos who have dominated the last five years, with 10 consecutive derby wins prior to the Copa del Rey final loss in May 2013.
Wednesday's meeting is the first of three matches between the two sides this month and, if Atletico avoids defeat in all three, it would put itself into a strong position in both competitions.
Simeone has Arda Turan available following injury, but David Villa has been ruled out after suffering a hamstring problem during Sunday's win over Sociedad.
Ancelotti is expected to name Iker Casillas in his side having used the 32-year-old throughout the Copa campaign, while Cristiano Ronaldo may play given he will miss the next league match due to suspension.
The Italian will also have Gareth Bale available after the Wales international returned to training on Monday following a leg injury.