Introduction Lipid metabolism is increasingly recognized as a critical factor in cancer progression, with lipid droplet accumulation emerging as a hallmark of aggressive cancers. However, the role of lipid hydrolysis—specifically the breakdown of stored lipids—in lung cancer remains underexplored. In this study, we aimed to investigate the involvement of lipid hydrolysis enzymes, particularly adipose triglyceride lipase (ATGL) and monoglyceride lipase (MGL), in non-small cell lung cancer (NSCLC). Methods Our initial investigations involved a comprehensive analysis of tumor and adjacent normal tissue from NSCLC patients using activity-based protein profiling (ABPP), shotgun proteomics, and lipidomics. Building on these findings, we explored the functional consequences of lipid hydrolase depletion by generating knockout models of ATGL and MGL in a panel of NSCLC cell lines. Results and Discussion We found significant downregulation of several lipid hydrolases, including MGL, in lung tumors compared to normal tissue. Lipidomic profiling revealed substantial triglyceride accumulation and elevated levels of ceramides and lysophosphatidylcholines in tumors, suggesting a disruption in lipid catabolism. Interestingly, we observed that the deletion of ATGL or MGL in NSCLC cell lines led to increased proliferation in some but not all cell lines, which correlated with elevated de-novo fatty acid synthesis. These results suggest that the downregulation of lipid hydrolases may contribute to cancer cell proliferation by altering lipid metabolism. To further elucidate these mechanisms, ongoing experiments are investigating nutrient utilization, lactate excretion, as well as mitochondrial respiration and glycolysis. Additionally, we are generating cell lines overexpressing lipid hydrolases that were found to be downregulated in tumors to assess reversibility of the observed metabolic changes and its effect on proliferation. Overall, this study underscores the significant role of lipid hydrolysis in lung cancer metabolism and progression. Innovative aspects • Direct measurement of lipid hydrolase activity in freshly excised lung tumors and paired normal tissues, providing immediate insights into enzyme functionality • Comprehensive combination of activity-based proteomics, shotgun proteomics, and lipidomics to create a holistic profile of lipid metabolism dysregulation in lung cancer • Application of cutting-edge mass spectrometry techniques to identify novel metabolic biomarkers and therapeutic targets directly from patient-derived samples