Updated project metadata. Heme is a cofactor with myriad roles and is essential to almost all living organisms. Accordingly, bacte-rial pathogens have developed numerous mechanisms to acquire heme from their hosts. Beyond classi-cal gas transport and catalytic functions, heme is increasingly appreciated as a tightly controlled signal-ling molecule regulating protein expression. However, heme acquisition, biosynthesis and regulation is poorly understood beyond a few model organisms, and the heme-binding proteome has yet to be fully characterised in bacteria. Yet as heme homeostasis is critical for bacterial survival, heme-binding pro-teins are promising drug targets. Herein we report a chemical proteomics method for global profiling of heme-binding proteins in live cells for the first time. Employing a panel of heme-based clickable and photoaffinity probes enabled the profiling of 32-54% of the known heme-binding proteomes in Gram-positive and Gram-negative bacteria, and revealed previously unknown potential heme interactors. This simple-to-implement profiling strategy could be interchangeably applied to different cell types and sys-tems and fuel future research into heme biology.