The duo has lost a race against time to be fit for Tuesday's encounter with the club confirming both players have been left out of the provisional matchday squad
Atletico goalkeeper Courtois has been suffering from back spasms, while attacker Villa limped out of the 4-0 victory over Real Sociedad over a week ago with a hamstring strain.
Dani Aranzubia, who featured in Atleti's shock 2-0 reverse to Almeria this weekend, will continue in goal on Tuesday, while Raul Garcia and Adrian are Diego Simeone's options up front with Diego Costa suspended.
Atletico has not been beaten at home this season, but faces an almost insurmountable task in retaining the title it won last year at the expense of its city rival. Atleti needs to overturn a 3-0 deficit from the first leg at the Santiago Bernabeu last week.
Atletico squad to face Madrid:
Goalkeepers: Aranzubia, Bono
Defenders: Godin, Alderweireld, Manquillo, Juanfran, Insua, Miranda
Midfielders: Mario Suarez, Koke, Raul Garcia, Arda Turan, Cebolla, Gabi, Diego, Sosa.
Strikers: Adrian, Mesa.