This study introduces FP-biotin, a biotinylated fluorophosphonate probe, as a powerful tool for activity-based profiling of serine hydrolases. The probe is designed to detect and visualize the dynamics of serine hydrolase expression and function in crude tissue extracts. FP-biotin reacts with serine hydrolases in an activity-dependent manner, allowing for the simultaneous monitoring of both protein function and expression. The probe was synthesized through a series of chemical reactions, including the conversion of 10-undecen-1-ol to iodinated compound 3, followed by the formation of a diethoxy phosphonate and subsequent conversion to an ethoxyhydroxy phosphonate. The final product, FP-biotin, was then coupled with a biotin tag through a long alkyl chain and two amide bonds. FP-biotin was tested with various serine hydrolases, including fatty acid amide hydrolase (FAAH), and demonstrated its ability to label these enzymes in an activity-dependent manner. The probe was also shown to label numerous serine hydrolases in rat tissues, with many displaying tissue-restricted expression patterns. The labeling was detected using SDS/PAGE-Western blotting, and the proteins were identified through mass spectrometry. The results indicated that FP-biotin can detect subnanomolar concentrations of serine hydrolases and distinguish between active and inactive forms of these enzymes. The study also demonstrated that FP-biotin can be used to profile the expression levels of serine hydrolases in cells, as shown by the detection of a strongly labeled 85-kDa protein in APH-transfected cells and a 65-kDa protein in FAAH-transfected cells. Additionally, FP-biotin was able to identify several candidate serine hydrolase activities with tissue-restricted patterns of expression in rat tissues, including a 33-kDa protein specifically expressed in prostate and a 42-kDa protein specifically expressed in testis. The findings highlight the utility of FP-biotin as a rapid and high-sensitivity probe for detecting serine hydrolase activities in crude cell and tissue samples. The probe's ability to detect both the functional state and expression level of these enzymes makes it a valuable tool for proteomics studies. The study also suggests that FP-biotin could be integrated with other techniques, such as MALDI mass spectrometry, to facilitate the molecular identification and functional comparison of serine hydrolases. Overall, the results demonstrate that FP-biotin is a powerful tool for the activity-based profiling of serine hydrolases.This study introduces FP-biotin, a biotinylated fluorophosphonate probe, as a powerful tool for activity-based profiling of serine hydrolases. The probe is designed to detect and visualize the dynamics of serine hydrolase expression and function in crude tissue extracts. FP-biotin reacts with serine hydrolases in an activity-dependent manner, allowing for the simultaneous monitoring of both protein function and expression. The probe was synthesized through a series of chemical reactions, including the conversion of 10-undecen-1-ol to iodinated compound 3, followed by the formation of a diethoxy phosphonate and subsequent conversion to an ethoxyhydroxy phosphonate. The final product, FP-biotin, was then coupled with a biotin tag through a long alkyl chain and two amide bonds. FP-biotin was tested with various serine hydrolases, including fatty acid amide hydrolase (FAAH), and demonstrated its ability to label these enzymes in an activity-dependent manner. The probe was also shown to label numerous serine hydrolases in rat tissues, with many displaying tissue-restricted expression patterns. The labeling was detected using SDS/PAGE-Western blotting, and the proteins were identified through mass spectrometry. The results indicated that FP-biotin can detect subnanomolar concentrations of serine hydrolases and distinguish between active and inactive forms of these enzymes. The study also demonstrated that FP-biotin can be used to profile the expression levels of serine hydrolases in cells, as shown by the detection of a strongly labeled 85-kDa protein in APH-transfected cells and a 65-kDa protein in FAAH-transfected cells. Additionally, FP-biotin was able to identify several candidate serine hydrolase activities with tissue-restricted patterns of expression in rat tissues, including a 33-kDa protein specifically expressed in prostate and a 42-kDa protein specifically expressed in testis. The findings highlight the utility of FP-biotin as a rapid and high-sensitivity probe for detecting serine hydrolase activities in crude cell and tissue samples. The probe's ability to detect both the functional state and expression level of these enzymes makes it a valuable tool for proteomics studies. The study also suggests that FP-biotin could be integrated with other techniques, such as MALDI mass spectrometry, to facilitate the molecular identification and functional comparison of serine hydrolases. Overall, the results demonstrate that FP-biotin is a powerful tool for the activity-based profiling of serine hydrolases.