Abstract The dynamic proteome plays numerous roles in the interactions of microbes—whether they are invading pathogens or symbiotic organisms—and their hosts. Host and microbe sense, respond, and manipulate each other's biology via a multitude of mechanisms, resulting in alterations in protein expression or posttranslational modification that influence protein localization, activity, or binding partners. The intrinsic, temporal, and spatial complexity of multispecies systems makes identifying the molecular players challenging. Chemical proteomic approaches apply small molecule chemical tools to interrogate protein function, interactions or modifications. This review highlights recent advances in the application of these methods at the host–microbe interface.