Alterations in lipid metabolism are a hallmark of cancer, with increased lipogenesis, fatty acid uptake, and lipid droplet accumulation emerging as defining features of many solid tumors. However, the role of lipid hydrolysis in cancer metabolism remains underexplored. To address this, we employed unbiased serine hydrolase-directed activity-based protein profiling of patient-derived lung tumors and adjacent normal tissue, combined with comprehensive proteomics and lipidomics analysis. Strikingly, several lipid hydrolases showed reduced activity and abundance in lung tumors, including monoglyceride lipase (MGL), epoxide hydrolase 1 (EPHX1), carboxylesterase 1 (CES1), neutral cholesterol ester hydrolase 1 (NCEH1), and patatin-like phospholipase domain-containing protein 6 (PNPLA6). Concomitantly, the lipid profile of tumors was altered, with many lipid species elevated compared to normal tissue. Triacylglycerols were especially enriched in tumors, particularly those containing long-chain and highly unsaturated fatty acids, suggesting lipid droplet accumulation. This was further supported by increased levels of the lipid droplet scaffolding protein Perilipin-2 (PLIN2), while ATGL - the main triacylglycerol hydrolase - was reduced in tumors, as confirmed by immunofluorescent staining. Lipid substrate assays of the downregulated hydrolases revealed that most of the lipid hydrolases reduced in lung tumors possess monoacylglycerol hydrolysis activity, amongst others. Consistent with this, several monoacylglycerol species and the overall lipid class were more abundant in tumors, corroborating decreased monoacylglycerol hydrolysis in lung tumors. To investigate the functional consequences of reduced lipid hydrolase expression, we generated NSCLC cell lines stably overexpressing ATGL, MGL, EPHX1, CES1, NCEH1, and PNPLA6. Overexpression led to reduced proliferation and, in some cases, caused a concurrent increase in fatty acid β-oxidation, a pathway found suppressed in tumor tissue. Lipidomic profiling of these cells is ongoing to identify specific lipid alterations associated with hydrolase overexpression. Together, our findings indicate that reduced lipid hydrolase activity is a feature of lung tumors that contributes to a reprogrammed lipid metabolism and altered lipid profile. This shift may support tumor growth by limiting fatty acid oxidation and associated oxidative stress.