Dysregulation of lipid hydrolases contributes to diseases such as obesity, cardiovascular and central nervous system disorders and metabolic syndromes. Lipase inhibitors are employed clinically to treat these conditions and as experimental tools to study the physiological and pathological roles of lipases. However, off-target effects in both research and therapy applications can compromise full target selectivity and confound outcomes. To address this, we applied competitive activity-based protein profiling (ABPP) to identify novel lipase inhibitor off-targets. Using a serine hydrolase-targeting probe in combination with inhibitors of the lipolytic enzymes Adipose Triglyceride Lipase (ATGL), Hormone Sensitive Lipase (HSL), Monoacylglycerol Lipase (MGL) or Lysosomal Acid Lipase (LAL), we analyzed LX-2 human hepatic stellate cells, murine liver tissue, murine primary hepatocytes as well as bone marrow-derived macrophages (BMDM). This method revealed several known as well as some unknown off-targets of HSL inhibitor Hi 76-0079, MGL inhibitors JZL-184 and Lu AG06466 as well as LAL inhibitors Lalistat-1 and Lalistat-2. We further observe differences in the lipase activity profiles between hepatocytes and BMDM. In conclusion, our competitive ABPP-based approach uncovers tissue-specific off-targets of prominent lipase inhibitors, highlighting their potential effects on cellular function and the importance of tissue context in lipase inhibition studies.