As amphipathic molecules, BAs have strong antibacterial effects, preventing overgrowth of the gut microbiota and defending the invasion of pathogens. However, some disease-causing pathogens can survive from the BA stress and knowledge is limited about how they develop BA tolerance. In the current study, we applied a quantitative chemoproteomic strategy to profile BA-interacting proteins in bacteria, aiming to discover the sensing pathway of BAs. By using the clickable and photo-affinity analogue BA probes with quantitative mass spectrometry, we identified 588 CA-interacting proteins and 435 DCA-interacting proteins.