In this study, we used a comprehensive approach involving endogenous peptidome along with bioinformatics analysis, to identify and evaluate potential biomarkers for HCC. Serum samples from 40 subjects, comprising 20 HCC cases and 20 patients with liver cirrhosis (CIRR), were analyzed. Among 2568 endogenous peptides, 68 showed significant differential expression between the HCC vs. CIRR. Further analysis revealed three endogenous peptides (VMHEALHNHYTQKSLSLSPG, NRFTQKSLSLSPG, and SARQSTLDKEL) that showed far better performance compared to AFP in terms of area under the receiver operating characteristic curve (AUC), showcasing their potential as biomarkers for HCC. Additionally, endogenous peptide IAVEWESNGQPENNYKT that belongs to precursor protein Immunoglobulin heavy constant gamma 4, was detected in 100% of the HCC group and completely absent in all samples of the CIRR group, suggesting a promising diagnostic biomarker. Gene ontology and pathway analysis revealed the potential involvement of these dysregulated peptides in HCC.