Lipids are crucial for maintaining membrane homeostasis and many other functions of healthy cells. Cancer and proliferating cells rely on a sufficient supply of lipids, making lipid metabolism a promising therapeutic target in cancer. Increased uptake and biosynthesis of lipids are frequently observed cancer phenotypes, and the accumulation of neutral lipids in lipid droplets has embarked as a hallmark of aggressive cancers. We aim to understand the relationship between lipid droplet accumulation and cancer by studying the role of intracellular lipid hydrolases and their influence on the metabolism of cancer cells in the context of non-small cell lung cancer (NSCLC). Tumors and paired normal tumor-adjacent (NTA) tissue of NSCLC patients were collected and subjected to in-depth proteomics, lipidomics, and activity-based protein profiling (ABPP) by state-of-the-art mass spectrometry techniques. ABPP identified several lipid hydrolases, including monoglyceride lipase (MGL), with decreased activity in tumors (fold change > 1.5) compared to paired NTA tissue. Proteomic profiling revealed distinct differences in lung tumor protein composition, enriched in DNA replication and ribosome proteins, while PPAR-α signaling and lipid catabolic enzymes were downregulated. Lipidomic analysis displayed elevated levels of triglycerides, monoglycerides, and ceramides in tumors, further supporting perturbation in lipid hydrolysis. Collectively, these data highlight the implication of lipid hydrolysis in NSCLC through downregulated lipid catabolism pathways. To further understand the functional consequences of lipid hydrolase modulation, we genetically manipulated a panel of NSCLC cell lines (A549, H1299, H358, H441) by deleting adipose triglyceride lipase (ATGL) or MGL. Lipase deletion led to increased LDs, altered proliferation, and metabolic changes in some NSCLC cell lines. Metabolic flux analysis unveiled a metabolic switch in lipase-deficient cell lines, which produce more glucose-derived fatty acids, irrespective of extracellular lipid availability. Our study delves into the intricate interplay between lipid hydrolysis enzymes, NSCLC cell metabolism, and proliferation. By unraveling these mechanisms, we aim to identify novel dependencies of lung cancer cells, providing insights into the drivers of lung cancer progression. This knowledge holds the potential to uncover new therapeutic targets and personalized treatment strategies for NSCLC.