This volume explores the cutting-edge of proximity-dependent protein biotinylation approaches, focusing on innovative biotin ligase (BioID) and peroxidase-based methods, like APEX. Spanning a diverse array of biological systems, including bacteria, zebrafish, plants, and mammals, this collection showcases methods that aid in the identification of protein interactions, localization, membrane topology, trafficking, and secretion dynamics. Additionally, the book features applications at the host/microbe interface and for the use of tissue-specific biotinylation for studying interorgan communication, as well as in silico validation methods. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, detailed lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and expert tips for troubleshooting and avoiding known pitfalls. Authoritative and practical, Proximity-Dependent Protein Biotinylation: Methods and Protocols serves as an essential guide for researchers advancing the study of proxeome biology.