Extracellular vesicles (EVs) are cell-derived particles carrying cytoplasmic content enclosed by a lipid bilayer and range in diameter between 30 nm and several μm. EVs are broadly classified based on their size, categorized into large EVs (L-EVs, 100–1000 nm) and small EVs (S-EVs, 30–150 nm). Collectively, EVs function in mediating cell-to-cell communication through their cargo, which includes specific proteins (signaling molecules, receptors, integrins, and cytokines), bioactive lipids, and nucleic acids (mRNAs, microRNAs, long noncoding RNAs, and DNA). Proteins on the surface of EVs can help regulate the interaction of EVs with their microenvironment, influencing EV function, distribution in the body, time in circulation, and pharmacokinetics. The EV surface protein network (surfaceome) acts as a fundamental signaling gateway by bridging intra- and extracellular signaling networks and is a key resource of potential circulating biomarkers of health and disease and therapeutic targets for enhanced delivery and retention. Here, we outline a systematic “how to” protocol (along with useful insights/tips) to label and capture the EV surface using membrane-impermeant derivative of biotin and obtain the EV surfaceome using mass spectrometry. Further, we have applied this approach to isolated EV subtypes (small and large EVs) to understand the surface proteome of different EVs.