This report summarises the presentations and activities of the SELECTBIO Workshop on Rigor and Reproducibility in EV Research and Single EV Analysis held in San Diego, USA, in December 2021. The motivation for the session was the recognition that progress in the extracellular vesicle (EV) field is limited by the availability of rigorous and reproducible EV measurement tools. These tools are absolutely required for EVs to evolve from a research lab curiosity to something that will improve our ability to understand, diagnose, treat, and prevent disease. The program focused on guidelines for EV measurement and characterization as laid out in the recent MISEV2018 and MIFlowCyt-EV publications, their implementation in routine practice, and their continued evolution as new EV measurement technologies are introduced. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.