Lecture about density gradient ultracentrifugation of extracellular vesicles (EVs) by An Hendrix as part of the third massive online open course (MOOC) of the International Society for Extracellular Vesicles (ISEV). The lecture covers the basic principles of density gradient ultracentrifugation, the performance of density gradient ultracentrifugation in terms of EV recovery and enrichment, practical information, do’s and don’ts as well as examples.