Introduction Serine hydrolases constitute a large and diverse family of enzymes that play pivotal roles in numerous physiological processes, including metabolism, signal transduction, and inflammation. These enzymes are characterized by a serine residue in their active site, which is crucial for their catalytic function. Serine hydrolases are involved in the hydrolysis of various substrates, making them essential for the regulation of lipid and carbohydrate metabolism, as well as protein processing. For instance, lipases, a subclass of serine hydrolases, are critical for the breakdown of triglycerides into free fatty acids and glycerol, which can then be used for energy production, especially during periods of fasting or increased energy demand. Other serine hydrolases contribute to lipid metabolism and the modulation of signaling pathways that govern energy balance. The regulation of serine hydrolase activity is highly tissue-specific, reflecting the distinct metabolic needs and functions of different organs. In the liver, serine hydrolases are involved in gluconeogenesis and the detoxification of xenobiotics, whereas in adipose tissue, they facilitate the release of stored fat. Muscle tissue relies on serine hydrolases for the rapid mobilization of energy substrates during physical activity, and the brain depends on these enzymes to maintain lipid homeostasis and neurotransmitter balance. This study aims to elucidate the effects of feeding state and dietary composition on serine hydrolase activity in key metabolic tissues of mice, providing insights into the dynamic and spatial regulation of energy mobilization and the potential for dietary interventions in metabolic health. Methods Fresh mouse tissues were incubated immediately after harvest with a serine hydrolase specific probe harboring a click moiety. This biorthogonal group was subsequently coupled to a biotin tag for enrichment of activity labeled enzymes on streptavidin beads. Enriched enzymes were subjected to label free mass spectrometry on a Bruker timsTOF operated in PASEF mode. To define a baseline of protein abundance, label free proteomics without enrichment was conducted additionally. Results and Discussion In this study, mice were subjected to different feeding regimens: fasting, regular feeding, and post fasting refeeding on either chow or high-fat diet. Samples from 12 individual tissues including liver, adipose tissue, brain, and muscle were collected and analyzed for serine hydrolase activities using activity-based protein profiling (ABPP). The results indicated significant variations in serine hydrolase activities depending on both the feeding state and diet. Fasting induced a marked increase in hydrolase activity in the liver and adipose tissues, while regular feeding maintained baseline activity levels. High-fat diet feeding resulted in altered activation of serine hydrolase activity dependent on fasting state. Surprisingly, even in brain we were able to pick up feeding dependent alterations in serine hydrolase activity. These findings underscore the dynamic and tissue dependent regulation of serine hydrolases by dietary inputs and highlight the potential for dietary interventions to modulate enzyme activity in specific tissues, with implications for metabolic diseases and therapeutic strategies. Innovative aspects • Feeding state dependent serine hydrolase activity profiles in 12 murine tissues • Cross tissue analysis of diet and feeding state impact