Bile acids (BAs) are a family of metabolites with an identical steroid core and versatile functional groups, which play central roles in digestion, metabolism and signaling. While endogenous receptors for selected BAs have been identified, a global BA-interactome map with site-specific resolution and ranked binding preferences is still lacking. Here, we report a high-throughput chemoproteomic platform named “SPIDER” that integrates ligand-directed release (LDR) chemistry with quantitative proteomics for site-specific and multiplexed profiling of BAs’ interactome. We applied SPIDER to quantify 770 interactions between six common BAs with 493 proteins in proteomes with both binding pockets mapped and binding preferences ranked. We discovered the microbiota-originated lithocholic acid (LCA) as an endogenous inhibitor of hydroxymethylglutaryl-CoA synthase 1 (HMGCS1), a key enzyme in the mevalonate pathway, and developed a potent covalent inhibitor for the enzyme based on an LCA-mimicking natural product to effectively lower nascent cholesterol biosynthesis. We further extended SPIDER to multiplexed interactome profiling of 19 FDA-approved drugs and established a comprehensive map of protein-drug interactions to guide drug screening and repurposing. These data highlight SPIDER as an enabling tool to streamline site-specific and comparative interactome profiling with diverse bioactive ligands in complex biological systems.