Recognition and eradication of infected cells by cytotoxic T lymphocytes is a key defense mechanism against intracellular pathogens. High-throughput definition of HLA class I-associated immunopeptidomes by mass spectrometry is an increasingly important analytical tool to advance our understanding of the induction of T cell responses against pathogens such as HIV-1. We utilized a liquid chromatography tandem mass spectrometry workflow including de novo-assisted database searching to define the HLA class I-associated immunopeptidome of HIV-1-infected human cells. We here report for the first time the identification of 75 HIV-1-derived peptides bound to HLA class I complexes that were purified directly from HIV-1-infected human primary CD4+ T cells and the C8166 human T cell line. Importantly, one third of eluted HIV-1 peptides had not been previously known to be presented by HLA class I. Over 82 of the identified sequences originated from viral protein regions for which T cell responses have previously been reported but for which the precise HLA class I binding sequences have not yet been defined. These results validate and expand the current knowledge of virus-specific antigenic peptide presentation during HIV-1 infection and provide novel targets for T cell vaccine development. This article is protected by copyright. All rights reserved.