Extracellular vesicles (EVs) are small derivatives of the cell that may mediate at least in part the effects of stem cells in vitro and in vivo. In light of strong interest in stem cell-based therapies, EVs are particularly attractive as therapeutic entities because they do not replicate and may have other safety advantages over whole stem cells. Here, we examine the current literature on stem cell-derived EVs, finding that differential centrifugation remains the most widely used central purification method for EVs. We then present a common variation of ultracentrifugation-based EV enrichment, followed by a protocol for determination of particle size and concentration by single particle tracking. Notes are provided to introduce technical variations and important considerations. Since rapid shifts in technique usage are expected to occur over the next several years, we briefly review new approaches to isolation and characterization.