Abstract: Hepatic lipotoxicity, driven by chronic exposure to excess fatty acids, represents a central pathogenic mechanism underlying a broad spectrum of metabolic liver disorders, most notably non-alcoholic fatty liver disease (NAFLD) and its progressive advanced forms. Palmitic acid (PA), the most abundant circulating saturated fatty acid in humans, has been widely used to induce lipotoxic stress in hepatocyte-derived cell models, including HepG2 cells. Despite the extensive use of this in vitro model, comprehensive and high-depth proteomic datasets capturing PA-induced cellular remodeling remain scarce. Such detailed molecular profiling is essential for elucidating the underlying biological mechanisms and for identifying high-confidence therapeutic targets. In this study, a comparative, multi-method sample preparation strategy prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was employed. The performance of three sample preparation strategies was systematically compared, including filter-aided sample preparation (FASP), solvent precipitation-based single-pot, solid-phase-enhanced sample preparation (SP4), and protein precipitation-based in-solution digestion. By integrating these approaches, the aim was to minimize methodological bias, enhance overall proteome coverage, and improve the robustness of differentially expressed protein (DEP) identification. HepG2 cells were treated with PA to induce lipotoxic stress, which was followed by label-free quantitative proteomic profiling using high-resolution mass spectrometry. Comparative analysis across the different preparation methods revealed substantial overlap in protein identifications, while each method uniquely contributed additional protein groups, collectively enabling deeper proteome coverage. Integration of the datasets significantly increased the confidence and robustness of DEP detection. Functional enrichment analysis demonstrated that PA treatment induced up-regulation of proteins involved in extracellular matrix organization, focal adhesion, complement and coagulation cascades, as well as fatty acid metabolism and peroxisome proliferator-activated receptor (PPAR) signaling pathways. These findings indicate extensive extracellular remodeling, inflammatory activation, and metabolic reprogramming in response to lipotoxic stress. In contrast, down-regulated proteins were predominantly enriched in ribosome biogenesis, translational machinery, and spliceosome-associated pathways, suggesting a global suppression of protein synthesis and RNA processing under sustained lipid overload. This study not only systematically identified and broadened the spectrum of potential therapeutic targets in a lipotoxic HepG2 cell model, but also uncovered novel molecular mechanisms related to transcription factor activation, organelle dysfunction, and calcium homeostasis dysregulation, thereby offering valuable data resources and mechanistic insights for studies on lipotoxicity and hepatotoxicity.