Proximity-dependent protein biotinylation is a transformative approach for studying bacterial surface proteomes during infection. By engineering a bacterial autotransporter to expose APEX2 on the bacterial surface, this protocol allows for the precise labeling and characterization of bacterial surface proteins, as well as host-derived bacterial-binding proteins (HBBPs) that interact with the pathogen during infection. The methodology integrates bacterial culturing, biotinylation, immunofluorescence labeling, and protein analysis to provide a comprehensive workflow for identifying key players in host–pathogen interactions. Insights gained from this approach can advance our understanding of bacterial virulence strategies and uncover potential therapeutic targets. Although the focus is on autotransporter and APEX2-based proximity labeling, the method can be adapted to other proximity labeling systems or tailored to specific bacterial species or infection models. By expanding the range of tools available to study microbial pathogenesis, surface-exposed proximity labeling opens new avenues for developing innovative strategies to combat infectious diseases.