Targeting cellular factors is considered a promising approach to combat viral infection and overcome the resistance issue associated with direct-acting antiviral (DAA) drugs administration. In the context of drug repurposing strategies, we have previously explored the antiviral efficacy of the MEK inhibitor zapnometinib against influenza virus and SARS-CoV-2. Herein, since zapnometinib was originally promoted as an anti-cancer drug, we aimed to investigate the impact of zapnometinib on the HLA class I ligandome post-treatment and IAV infection. By implementing the immunopeptidomics approach, the influence on the HLA-I allotype distribution of the HLA ligands was observed to coincide with alteration in the relative abundance of HLA-I-presented peptides. Furthermore, functional annotation analysis of the corresponding source proteins of presented ligands revealed a remarkable gene enrichment of distinct cellular processes. Taken together, zapnometinib and IAV showed a remarkable effect on the HLA-I ligandome plasticity as well as quantitative alterations in HLA ligandome composition.