Abstract MHC class I is required for antigen presentation to CD8 T cells. These cell-surface proteins are heterodimers consisting of a variable alpha chain associated with β2 microglobulin and are expressed on nucleated cells at varying levels. The antigen specificity of the interaction between MHC, peptide and T cell receptor can be exploited in development of personalized cancer vaccines and other immunotherapies against cancer. MHC sequences are species-specific and there are many different haplotypes of MHC class I. For this reason, the antibodies used for immunoprecipitation of MHC complexes are a critical component of the workflow for identification of antigens presented by MHC. Subclasses of mouse MHC class I are divided into classical (H-2D, H-2K and H-2L) and non-classical (H-2Q, H-2M and H-2T), and haplotypes of each are known for homozygous inbred laboratory strains of mice. One common strain is C57BL/6, which expresses H2-Db and H2-Kb classical MHC molecules. Antibody clone M1/42, which reacts with H-2 expressed on nucleated cells from mice of a, b, d, j, k, s and u haplotypes, is commonly referred to as a pan-MHC class I antibody. Antibody clones Y-3 and HB-27 recognize H2-Kb and H2-Db haplotypes, respectively. In this study we aimed to compare different antibodies to mouse MHC class I complexes for their efficiency of recovery of associated peptides. Input was murine EL-4 cells which are T lymphoblast cells derived from a C57BL/6 background. We performed immunoprecipitation with different antibodies either singly or sequentially to enrich MHC class I, followed by gentle elution of associated peptides and finally peptide analysis by mass spectrometry. Each immunoprecipitation workflow yielded a different (but overlapping) cohort of peptides, suggesting optimal methodologies for analyses of immunopeptidomes from common laboratory mouse strains. Citation Format: Seeta Nyayapathy, Richard Jones, Michael Ford, Michael Pisano, Julie M. Rumble. Immunopeptidome analysis of mouse MHC Class I [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1384.