Alterations in lipid metabolism are a hallmark of cancer that is ultimately determined by changes at the level of enzyme activity, which is often poorly reflected by transcript or protein abundance. The reliable and unbiased measurement of enzyme activities in tumors remains challenging. Activity-based protein profiling of serine hydrolases provides a powerful tool for assessing the activities of key lipid metabolizing enzymes and was used in this study to investigate lipid metabolism in fresh tumors and matched normal tissue of non-small cell lung cancer (NSCLC) patients. Together with comprehensive lipidomics analysis, this approach revealed extensive remodeling of the lipidome in lung tumors, characterized by accumulation of polyunsaturated fatty acid-containing triacylglycerols, and ether-linked lipids. Contrary to findings in other aggressive cancers, we observed a general downregulation of lipid hydrolase activity, including enzymes involved in mono- and triacylglycerol catabolism. This reduction in lipolytic activity was reflected by the lipidomic profiles and was associated with increased lipid droplet accumulation, suggesting a shift toward lipid storage and away from lipid catabolism in the tumors. Furthermore, when stratified based on 5-year recurrence follow up, primary tumors from patient who relapsed demonstrated greater impairment of neutral lipolysis compared to non-relapsed ones. Substrate profiling of overexpressed and purified lipases found deregulated in tumor vs. healthy tissue confirmed their preferences towards distinct lipid classes, especially mono- and triacylglycerols. Overexpression of selected lipid hydrolases in NSCLC cell lines resulted in altered lipid composition, impaired proliferation and triggered apoptosis, at least partially due to redox disbalance. These findings suggest that NSCLC cells reprogram lipid metabolism to favor storage over oxidation as a protective strategy against oxidative stress, highlighting the reactivation of lipid hydrolysis and fatty acid oxidation as a potential metabolic vulnerability in lung cancer.