Purpose <p>Entamoeba histolytica is the causative agent of amoebiasis or amoebic dysentery, remains a significant global health burden, particularly in regions with inadequate sanitation. Despite the availability of a reference genome, a substantial proportion of predicted genes remain annotated as hypothetical proteins or lack experimental validation. Limitations inherent to automated gene prediction pipelines along with the parasite's highly AT-rich, repetitive genome architecture have resulted in numerous coding regions unresolved, underscoring the need for experimental approaches to refine genome annotation. </p> Methods <p>We undertook a comprehensive proteogenomic analysis by searching publicly available high-resolution proteomic datasets from E. histolytica HM-1:IMSS trophozoites against a custom six-frame translated genome database. The proteomic data was searched using Proteome Discoverer suite at 1% false discovery rate. </p> Results <p>Many unique peptides were identified during database dependent searches and of these, 480 peptides did not map to the reference protein database and were referred to as genome search-specific peptides (GSSPs). Mapping these GSSPs to the genome supported the identification of 41 novel protein-coding genes and corrected annotation of 18 currently annotated genes. Conserved domain analysis revealed that 78% of novel proteins harboured identifiable functional domains, including TLDc domain-containing proteins and rhomboid peptidase family proteins. Comparative analysis confirmed conserved orthologs across related Entamoeba species.</p> Conclusion <p>This is the first study to demonstrate proteogenomic integration of peptide-level evidence with genomic data substantially improves genome annotation in E. histolytica, expanding its known coding repertoire and providing functional insights into previously uncharacterized regions with implications for understanding parasite biology and virulence mechanisms.</p>