The identification of peptides presented by HLA class I molecules to CD8+ T cells – i.e. the HLA-I immunopeptidome - is a useful tool to fish epitopes suitable for vaccinations and immunotherapies against cancer, infection and autoimmunity. These peptides are mainly generated by proteasomes through peptide hydrolysis and peptide splicing. Post-translationally spliced epitopes can enlarge the antigenic landscape and thus immunotherapy efficacy. Their identification is challenging, and the few methods proposed so far hinted toward a controversial broad spectrum of cis-spliced peptide frequencies in HLA-I immunopeptidomes. We here developed a new method named SPI-ART, which can identify cis-spliced peptides in HLA-I immunopeptidomes with precision and recall outperforming all other methods. The benchmark of the methods’ performances provided the proper analytical framework to explain the discordant results published so far. It also allowed to estimate the frequency of cis-spliced peptides in HLA-I immunopeptidomes, and to shed light on the biochemistry of these unconventional peptides and their potential antigenicity.