Levante will take its appeal against Copa del Rey elimination to the Court of Arbitration for Sport (CAS) after its initial move was rejected.
The LaLiga club alleged Barcelona used an ineligible player in the first leg of the last-16 tie, but its appeal was rejected by the Royal Spanish Football Federation (RFEF).
Levante beat Barca 2-1 at home in the first part of its 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, which Levante believed to have been suspended for the match after accumulating five yellow cards for Barca's 'B' team in the third tier.
Barca was 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 its complaint being lodged too late.
But Levante announced on Monday it would take its 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 quarterfinals has already been made, with Barcelona set to play Sevilla in the first leg of its tie on Wednesday.