Hepatocellular carcinoma (HCC) carries a poor prognosis and poses a serious threat to global health. Although early diagnosis and treatment can significantly reduce mortality, biomarkers of early HCC are lacking. This pilot study used 4D label-free quantitative proteomics to compare the proteomes of HCC and adjacent non-tumor tissue. A total of 66,075 peptides, 6,363 identified proteins, and 772 differentially expressed proteins (DEPs) were identified in specimens from three HCC patients. Through functional enrichment analysis of DEPs by Gene Ontology, KEGG pathway, and protein domain, we identified proteins with similar functions. Twelve DEPs (RPL17, RPL27, RPL27A, RPS5, RPS16, RSL1D1, DDX18, RRP12, TARS2, YARS2, MARS2, and NARS1) were selected for identification and validation by parallel reaction monitoring. Subsequent western blotting confirmed overexpression of RPL27, RPS16, and TARS2 in HCC compared to non-tumor tissue in eight pairs of clinical samples. Analysis of The Cancer Genome Atlas datasets associated increased expression of these proteins with poor prognosis. These data suggest that RPL27, RPS16, and TARS2 play an important role in HCC progression and may be biomarkers of overall survival in HCC patients.