Since the initial use of BirA* for proximity-dependent biotin identification (BioID), researchers have explored variations of, and alternatives to, this specific biotin ligase enzyme. The application of these biotin ligase modules across diverse model systems has shown advancements in biotinylation efficiency and reduced labeling time. However, these improvements have also introduced challenges, such as increased background labeling. Bacterial BioID, a relatively recent development, is currently limited to studying bait proteins using miniTurbo- and TurboID-based fusions. So far, a comprehensive comparative analysis of the various promiscuous biotin ligases (PBLs) has been reported for only one bacterial species. In this chapter, we present a practical guide for selecting the most effective PBL tailored to the specific requirements of the bait and its corresponding model system for BioID-based interactome mapping in bacteria. Using the Salmonella enterica serovar Typhimurium type III effector protein SopB as a case study, we highlight a pioneering exploration of endogenous effector interactions directly within bacterial cells.