Updated project metadata. Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed inside the cell to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of 315 overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes for the generation ofthat generate antigenic peptides, namely intracellular aminopeptidases ERAP1, ERAP2 and IRAP. Each All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with marked differences corresponding to distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 was largely highly destructive and greatly reduced limited peptides the variability of peptide sequences availableproduced for HLA-binding. Less than 710% of computationally predicted epitopes were found to be produced by enzymatic trimming experimentally, suggesting that proteolytic aminopeptidase processing may constitute a significant filter to successful epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness of predicting for viral antigenic epitopes.