Activity-based proteomic profiling (ABPP) enables the functional study of enzymes by employing small molecule probes that bind covalently to the active site of an enzyme. Activity-based probes can pene-trate cells and tissues and thereby allow enzymes to be targeted/labeled in their native state. Probes can be designed to target individual enzymes or whole enzyme groups, which makes ABPP a versatile protein profiling technique. In competitive ABPP, these small molecule probes compete with endoge-nous substrates for the enzyme's active site by displacing the substrate and blocking the active site terminally by covalently binding to it. Lipid hydrolases are essential enzymes that play a pivotal role in maintaining healthy cellular function by breaking down complex lipids into simpler components, and their dysregulation can contribute to a range of diseases, including obesity, cardiovascular disorders, and metabolic syndromes. Lipase inhib-itors serve as valuable tools for unraveling the intricate roles of lipases in diverse diseases, enabling researchers to manipulate lipid metabolism and study the downstream effects on cellular pathways and physiological processes. The application of lipase inhibitors in studies could present difficulties due to the possible engagement of various off-target effects on different enzymes, potentially leading to am-biguous outcomes and a lack of specificity toward a particular enzyme. We therefore set out to employ competitive ABPP to identify novel lipase inhibitor off-targets by combin-ing the use of a small molecule probe targeting serine hydrolases together with treatment of inhibitors against the lipolytic enzymes Adipose Triglyceride Lipase (ATGL), Hormone Sensitive Lipase (ATGL), Monoacylglycerol Lipase (MGL) or Lysosomal Acid Lipase (LAL) in murine primary hepatocytes as well as in bone marrow-derived macrophages (BMDM). With this method, we identify several known as well as some unknown off-targets for MGL inhibitor JZL-184 and LAL inhibitors Lalistat-1 and Lalistat-2. We further observe differences in the lipase activity profiles between hepatocytes and BMDM. In conclusion, our comprehensive approach utilizing competitive ABPP uncovers tissue-specific off-tar-gets of prominent lipase inhibitors, shedding light on their potential implications for cellular function and underscoring the significance of considering tissue variability when studying lipase inhibition effects.