The row centres on an incident early in the second half when Barcelona appealed for a penalty after Atletico goalkeeper Juan Musso appeared to have put the ball back into play from a goal kick before defender Marc Pubill handled it inside the six-yard box to retake it.
Referee Istvan Kovacs waved play on , and the VAR team did not summon him to the monitor, drawing furious reactions from the Barcelona bench.
"Barcelona inform that the club's legal services have submitted a complaint to UEFA today regarding the events in the Champions League quarter-final first leg against Atletico Madrid," the club said in an official statement on Thursday.
"The complaint centres around a specific action. In the 54th minute of the match, after play had been restarted correctly, an opponent player picked up the ball in their area without being shown the corresponding penalty," it added.
"Barcelona understand that this decision, along with a grave lack of intervention by VAR, represents a major error.
"Accordingly, the club has requested that an investigation be opened, access to refereeing communications, and, where applicable, official acknowledgement of the errors and the adoption of the relevant measures."
Interpretations of similar incidents have varied. In the Champions League quarter-finals in April 2024, the referee did not award a penalty to Bayern Munich after Arsenal defender Gabriel Magalhaes touched the ball following what appeared to be a restart from goalkeeper David Raya.
Later in 2024, VAR intervened to award Club Brugge a penalty in a comparable exchange involving Aston Villa goalkeeper Emiliano Martinez and defender Tyrone Mings.