Neymar was handed the punishment for sarcastically applauding the fourth official after being shown a second yellow card in the 2-0 defeat at Malaga earlier this month.
The Brazilian forward's show of dissent earned him an additional two-match suspension on top of the one-game ban that came as a result of the red card at La Rosaleda, ruling him out of the clash with Madrid at Santiago Bernabeu this weekend and the meeting with Osasuna next week.
Barca argued it could not be proved Neymar's reaction was directed at the official, but the Royal Spanish Football Federation's Appeals Committee rejected their claim and upheld the sanction.
The Blaugrana, however, could yet take their appeal to the Court of Arbitration for Sport in order to have his punishment overturned.
It was the second successive season Luis Enrique's charges have fallen short in the last eight, after going down to Atletico in 2015-16.
Now, the Clasico takes on added importance for the club as they seek to keep their slim Liga hopes alive.
Barca go into Sunday's game three points behind Madrid, who also hold a game in hand over their rivals.
Defeat at the weekend would effectively hand the title to the Merengue for the first time since 2011-12, leaving the Copa del Rey, where they will meet Alaves in May's final, as their last opportunity for major silverware this season.