The article describes the development and application of a biotinylated fluorophosphonate (FP-biotin) as a probe for visualizing serine hydrolases in crude tissue and cell extracts. Serine hydrolases play crucial roles in various biological processes and diseases, but their functions are often not well understood due to the lack of effective methods for studying them. FP-biotin is designed to react specifically with active serine hydrolases, allowing for the detection of their activity and expression levels. The authors demonstrate that FP-biotin can label multiple serine hydrolases in a tissue-specific manner, with some proteins showing tissue-restricted patterns of expression. Kinetic analyses reveal that the labeling rates vary among different serine hydrolases, providing insights into their functional states. The method is also shown to be sensitive enough to detect subnanomolar concentrations of serine hydrolases and to monitor changes in their expression levels. Overall, FP-biotin offers a powerful tool for profiling serine hydrolases in complex biological samples, facilitating the identification of potential pharmaceutical targets and the understanding of their roles in physiological and pathological processes.The article describes the development and application of a biotinylated fluorophosphonate (FP-biotin) as a probe for visualizing serine hydrolases in crude tissue and cell extracts. Serine hydrolases play crucial roles in various biological processes and diseases, but their functions are often not well understood due to the lack of effective methods for studying them. FP-biotin is designed to react specifically with active serine hydrolases, allowing for the detection of their activity and expression levels. The authors demonstrate that FP-biotin can label multiple serine hydrolases in a tissue-specific manner, with some proteins showing tissue-restricted patterns of expression. Kinetic analyses reveal that the labeling rates vary among different serine hydrolases, providing insights into their functional states. The method is also shown to be sensitive enough to detect subnanomolar concentrations of serine hydrolases and to monitor changes in their expression levels. Overall, FP-biotin offers a powerful tool for profiling serine hydrolases in complex biological samples, facilitating the identification of potential pharmaceutical targets and the understanding of their roles in physiological and pathological processes.