The gut microbiome plays an important role in maintaining human health through participating in the production of metabolites. Bile acids (BAs) are a group of diet-derived metabolites that are produced by the host and processed by the gut microbiome, exerting significant effects on food digestion, metabolism homeostasis, and immune regulation. The glyco- or tauro- conjugated bile acids/bile salts are secreted into the small intestine, and subjected to bio-transformations to afford secondary BAs and other BA-derivatives. BA metabolism relies on the enzymes generated in gut microbiota, of which bile salt hydrolases (BSHs) catalyze the hydrolysis of conjugated bile salts to make free BAs. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Herein, we proposed an array of activity based probes (ABPs), employing two distinct warheads: acyloxymethyl ketone (AOMK) and fluoromethyl ketone (FMK). The AOMK probes are used to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms. By employing the FMK probes, along with a systems biochemistry approach, we have identified gut microbiota-associated BSHs that exhibit distinct substrate preferences, revealing that different microbes contribute to the diversity of the host bile acid pool. We envision that this chemoproteomic approach will reveal how secondary bile acid metabolism controlled by BSHs contributes to the etiology of various inflammatory diseases. Taken together, these works highlight the utility of BSH-targeted ABPs to profile activity alternations of BSHs and their substrate preference.