This study aims to compare the proteomic profiles of extracellular vesicles (EVs) isolated from four human liver cell lines—THLE-2 (non-tumorigenic immortalized hepatocytes), HepG2, HEP3B, and Huh7 (hepatocellular carcinoma cell lines)—using two different isolation methods: PHS capture and ultracentrifugation (UC). The goal is to identify method-dependent and cell line-specific protein signatures in EVs, with particular focus on tumor-associated biomarkers. Mass spectrometry-based proteomic analysis was performed to quantify protein expression levels across all samples. Differential expression analysis revealed distinct protein profiles between the four cell lines, as well as differences attributable to the isolation methods employed. Key tumor-associated biomarkers are highlighted in the dataset for further investigation. This dataset provides a valuable resource for understanding EV protein cargo heterogeneity in liver cancer and for evaluating the performance of PHS capture as an alternative EV isolation technique compared to the conventional UC method. The raw mass spectrometry data and protein identification results have been submitted to iProX in accordance with ProteomeXchange data sharing policies.