Lipids are crucial for maintaining membrane homeostasis and many other functions of healthy cells. A sufficient supply of lipids is essential for rapidly proliferating cells. Cancer cells frequently show increased uptake of lipids, making lipid metabolism a promising therapeutic target in cancer. More recently, the accumulation of neutral lipids like triacylglycerol and cholesterol esters in lipid droplets has evolved as a hallmark of aggressive cancers. In addition to increased uptake and biosynthesis of lipids, a decrease in intracellular lipid hydrolysis contributes to lipid droplet accumulation. However, lipid hydrolases' role in the cancer context is less widely explored. By employing state-of-the-art mass spectrometry techniques, we aimed to explore lipid hydrolysis in non-small cell lung cancer (NSCLC) on the proteome, lipidome, and serine hydrolase activity level. Tumors and normal tumor-adjacent (NTA) tissue of NSCLC patients were collected and subjected to in-depth proteomics, lipidomics, and activity-based proteomic profiling (ABPP). Tissues were collected immediately after tumor resection and flash-frozen. In the case of ABPP, tissues were immediately incubated with a small serine hydrolase-specific probe recognized by active serine hydrolases. Labeling active serine hydrolases with the probe allowed us to enrich active enzymes belonging to this class of enzymes (including lipases) and subsequently quantify the abundance of active enzymes. Through ABPP, we identified several serine hydrolases with higher activities (fold change > 1.5) in healthy tissue, including monoglyceride lipase (MGL), neuropathy target esterase (PNPLA6), epoxide hydrolase (EPHX1), neutral cholesterol ester hydrolase (NCEH1) and liver carboxylesterase 1 (CES1). Proteomics analysis revealed that several of those lipid hydrolases, as well as several fatty acid-binding proteins, are also higher in abundance in NTA tissue. Furthermore, lipidomics analysis on the same tissues exposed significant triglyceride accumulation and higher levels of ceramides and lysophosphatidylcholines in tumors. Collectively, these data highlight the implication of lipid hydrolysis in NSCLC and suggest that lung cancer cells shut down lipid catabolism pathways contributing to the observed changes in the lipidome.