Lipid hydrolases are essential regulators of lipid metabolism and their dysregulation can contribute to a range of diseases, including obesity, cardiovascular and central nervous system disorders as well as metabolic syndromes. Lipase inhibitors are used in clinical development to treat several of these conditions and serve as valuable research tools to study the role of lipases in diverse diseases, enabling researchers to dissect lipid metabolic pathways and study the downstream effects on physiological processes. However, off-target effects in both applications can compromise full target selectivity and confound both experimental results and therapeutic outcomes. To address this, we employed competitive activity-based protein profiling (ABPP) to identify novel lipase inhibitor off-targets across different tissues. In competitive ABPP, small molecule probes compete with endogenous substrates by covalently binding to the enzyme's active site and terminally blocking it. Our approach combines a serine hydrolase-targeting probe with inhibitor treatment against the lipolytic enzymes Adipose Triglyceride Lipase (ATGL), Hormone Sensitive Lipase (HSL), Monoacylglycerol Lipase (MGL) or Lysosomal Acid Lipase (LAL) in LX-2 human hepatic stellate cells, murine liver tissue, murine primary hepatocytes as well as in murine bone marrow-derived macrophages (BMDM). With this method, we identify several known as well as some unknown off-targets for MGL inhibitors JZL-184 and Lu AG06466, LAL inhibitors Lalistat-1 and Lalistat-2, as well as HSL inhibitor Hi 76-0079. We further observed differences in the lipase activity profiles between hepatocytes and BMDM. The identified off-targets were validated via over-expression in Expi293F cells and subsequent activity-based gel electrophoresis. In conclusion, our comprehensive approach utilizing competitive ABPP uncovers tissue-specific off-targets of prominent lipase inhibitors used in lipolytic enzyme research as well as in clinical development, shedding light on their potential implications for therapeutic effect as well as cellular function and underscoring the significance of considering tissue variability when studying lipase inhibition effects.