Research on extracellular vesicles (EVs) is growing exponentially due to an increasing appreciation of EVs as disease biomarkers and therapeutics, an expanding number of EV-containing materials under study, and application of new preparation, detection, and cargo analysis methods. Diversity of both sources and methodologies imposes challenges on the comparison of measurement results between studies and laboratories. While reference guidelines and minimal requirements for EV research have achieved the important objective of assembling community consensus, it is also essential to understand which methodologies and quality controls are currently being applied, and how usage trends are evolving. As an initial response to this need, the International Society for Extracellular Vesicles (ISEV) performed a worldwide survey in 2015 on “Techniques used for the isolation and characterization of extracellular vesicles” and published the results from this survey in 2016. In 2019, a new survey was performed to assess the changing state of the field. The questionnaire received more than 600 full or partial responses, and the present manuscript summarizes the results of this second worldwide survey. The results emphasize that separation methods such as ultracentrifugation and density gradients are still the most commonly used methods, the use of size exclusion chromatography has increased, and techniques based on tangential flow and microfluidics are now being used by more than 10% of respondents. The survey also reveals that most EV researchers still do not perform sample quality controls before or after isolation of EVs. Finally, the majority of EV researchers emphasize that separation and characterization of EVs should receive more attention.