Levante will take their appeal against Copa del Rey elimination to the Court of Arbitration for Sport (CAS) after their initial move was rejected.
The LaLiga club alleged Barcelona used an ineligible player in the first leg of the last-16 tie, but their appeal was rejected by the Royal Spanish Football Federation (RFEF).
Levante beat Barca 2-1 at home in the first part of their two-legged clash, as Ernesto Valverde shuffled his pack, giving games to fringe players and youngsters.
Among the unfamiliar names in the starting XI was defender Chumi, who Levante believed to have been suspended for the match after accumulating five yellow cards for Barca's 'B' team in the third tier.
Barca were convinced Chumi was eligible, with Valverde suggesting the defender's suspension was active for Sunday's win over Eibar, while the club understood a rule change in November meant a reserve player can feature for the first team in another competition as long as the suspension is less than a two-match ban.
However, the RFEF ultimately rejected Levante's appeal due to their complaint being lodged too late.
But Levante announced on Monday they would take their appeal to the CAS.
"Levante will file an appeal with the Court of Arbitration for Sport," the club wrote on Twitter.
The draw for the quarter-finals has already been made, with Barcelona set to play Sevilla in the first leg of their tie on Wednesday.
