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Here, we develop PEPSeek, a web-server based software to allow higher performance in the identification of pathogen-derived epitope candidates detected via mass spectrometry in MHC class I immunopeptidomes. We apply it to human and mouse cell lines infected with either SARS-CoV-2, Listeria monocytogenesor Chlamydia trachomatis, thereby identifying a large number of novel antigens and epitopes that we prove to be recognized by CD8+T cells. In infected cells, we identified antigenic peptide features that suggested how processing and presentation of pathogenic antigens differ between pathogens. The quantitative tools of PEPSeek also helped to define how C. trachomatisinfection cycle could impact on the antigenic landscape of the host human cell system, likely reflecting metabolic changes occurred in the infected cells.