Valencia have confirmed that Nani will be forced to miss Sunday's LaLiga meeting with champions Barcelona.
The Portugal international has been sidelined since suffering a hamstring injury during the 2-1 victory over Real Madrid on February 22.
It was hoped that Nani - who has yet to resume full training - would be available to face Luis Enrique's side, but the former Manchester United winger has suffered a setback.
"During the process of his recovery from an injury to his left thigh, Nani has suffered an overload in his right adductors, which rules him out of being available for the next match," the club said in a statement.
Forward Rodrigo, who broke his ankle in January and is expected to be out for the rest of the season, was able to complete running drills on Tuesday for the first time since sustaining the injury.