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Summary: Plasma extracellular vesicles (EVs) represent a potential resource for biomarkers of multiple diseases. Here, we present a protocol for obtaining EVs from human plasma using asymmetrical flow field-flow fractionation technology. We describe steps for using tandem mass tags to perform comparative proteomic studies of a large clinical cohort. We then detail targeted quantitative analysis of differential proteins based on a parallel reaction monitoring technique.For complete details on the use and execution of this protocol, please refer to Wu et al. (2020)1 and Li et al. (2023).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.