Objective: Antigen-specific immunotherapy is a promising strategy to treat hepatitis B virus (HBV) infection and (HBV-related) hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by HLA-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumor associated antigens (TAAs) to guide development of antigen-specific immunotherapy. Design: Primary human hepatocytes were isolated with high purity from (HBV infected) non-tumor and HCC tissues using a newly designed perfusion-free procedure. Subsequently, hepatocyte-derived HLA-bound peptides were identified by mass spectrometry after which source proteins were subjected to gene ontology and pathway analysis. Finally, all HBV-antigen and TAA-derived HLA-peptides were extracted and a selection was tested for immunogenicity. Results: We acquired a high quality HLA-I peptidome of 2x105 peptides, of which source proteins were associated with hepatocyte function. Importantly, we demonstrated HLA-I presentation of HBV-derived and TAA-derived peptides for the first time in immune cell-depleted primary liver cell isolates. The peptidome included 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAAs that were exclusively identified in tumor eluates. Of these, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides. Conclusion: We present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Our results directly aid development of antigen-specific immunotherapy for HBV infection and HCC. Described methodology can also be applied to personalize immunotherapeutic treatment of liver diseases in the future.