Lipids are crucial for maintenance of membrane homeostasis and many other functions of healthy cells and sufficient supply of lipids is especially important for cells that are rapidly proliferating. Cancer cells frequently show increased uptake of lipids as well as increased rate of lipogenesis, making lipid metabolism a promising therapeutic target in cancer. More recently, 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, decrease in intracellular lipid hydrolysis contributes to lipid droplet accumulation. However, the role of lipid hydrolases in the context of cancer 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 adjacent (healthy) 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, or in the case of ABPP immediately incubated with a small serine hydrolase-specific probe that is 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. By ABPP we identified a number of 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 a number of fatty acid binding proteins are also higher in abundance in the healthy tissue. Furthermore, a number of proposed PPARalpha targets are downregulated in tumor tissue, and lipidomics analysis on the same tissues exposed significant triglyceride accumulation as well as 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. We currently further investigate the role of individual lipid hydrolases and their regulators on metabolism, proliferation, migration and invasion of different cancer cell lines under different growth conditions to obtain more mechanistic insights.