Extracellular vesicles (EVs) have emerged as a novel class of regulators of tissue homeostasis, mediating inter-tissue communication through the exchange of bioactive molecular cargoes. However, progress in this field has been hindered by the lack of robust techniques for labeling tissue-specific EVs and tracking their biodistribution and cellular uptake, while minimally disturbing their biochemical and functional properties. Recent advancements in bioorthogonal chemistry and click reactions offer a promising solution by enabling specific labeling of EV surface components, including proteins, saccharides, and lipids. This review summarizes the latest literature on these approaches, highlighting their potential for precise EV identification, isolation, and targeted delivery in vivo. These methodologies are expected to significantly enhance our understanding of the role of EVs in tissue homeostasis and their applications in diagnostics and therapy.