Extracellular vesicles (EVs) are membrane-enclosed vesicles released by cells. They carry proteins, nucleic acids, and metabolites which can be transferred to a recipient cell, locally or at a distance, to elicit a functional response. Since their discovery over 30 years ago, the functional repertoire of EVs in both physiological (e.g., organ morphogenesis, embryo implantation) and pathological (e.g., cancer, neurodegeneration) conditions has cemented their crucial role in intercellular communication. Moreover, because the cargo encapsulated within circulating EVs remains protected from degradation, their diagnostic as well as therapeutic (such as drug delivery tool) applications have garnered vested interest. Global efforts have been made to purify EV subtypes from biological fluids and in vitro cell culture media using a variety of strategies and techniques, with a major focus on EVs of endocytic origin called exosomes (30-150 nm in size). Given that the secretome comprises of soluble secreted proteins, protein aggregates, RNA granules, and EV subtypes (such as exosomes, shed microvesicles, apoptotic bodies), it is imperative to purify exosomes to homogeneity if we are to perform biochemical and biophysical characterization and, importantly, functional dissection. Besides understanding the composition of EV subtypes, defining molecular bias of how they reprogram target cells also remains of paramount importance in this area of active research. Here, we outline a systematic "how to" protocol (along with useful insights/tips) to obtain highly purified exosomes and perform their biophysical and biochemical characterization. This protocol employs a mass spectrometry-based proteomics approach to characterize the protein composition of exosomes. We also provide insights on different isolation strategies and their usefulness in various downstream applications. We outline protocols for lipophilic labeling of exosomes to study uptake by a recipient cell, investigating cellular reprogramming using proteomics and studying functional response to exosomes in the Transwell-Matrigel™ Invasion assay.