The striker was booked for diving last weekend when Atletico drew 2-2 with Real and the authorities have upheld the referee's original decision.
Diego Costa will serve a one-match Liga ban after his yellow card during the Madrid derby was upheld by the Spanish Football Federation (RFEF).
The Atletico Madrid forward was booked in the 64th minute of Sunday's 2-2 draw at the Vicente Calderon after the referee deemed him to dive under a challenge from Real Madrid defender Alvaro Arbeloa.
The Rojiblancos appealed the decision made by referee Carlos Delgado, but the RFEF ruled Friday that the caution was justified, which means Costa will now miss Saturday's clash with Celta Vigo as it was his fifth yellow of the season.
The striker, who made his Spain debut against Italy on Wednesday, will also have to pay a fine of 800 euro while Atletico will face a 350 euro charge.
"In the full viewing of the play in its various outlets it is not apparent that the cause of the fall of the player is contact with the opponent," a league statement read. "So it does not disprove the contents of the referees report when he says the player simulated a foul.
"Accordingly the claims are dismissed, confirming the warning and disciplinary consequences."
Costa will not be the only player unavailable for Atletico coach Diego Simeone on Saturday, with Diego Godin and Arda Turan also serving one-match bans following bookings against Real.