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Typically, the analysis of EV\u2010associated biomarker characteristics such as size and surface markers requires pre\u2010purification from large volumes of complex biofluids, leading to longer turnaround times and more technical variability. To overcome these limitations, we developed a one\u2010step workflow that combines size\u2010based separation with size and surface marker characterisation of EVs in minute volumes from different biological fluids. We coupled a multi\u2010angle light scattering detector (MALS) and a fluorescent light detector (FLD) in\u2010line with the asymmetrical flow field\u2010flow fractionation (AF4) equipment. The AF4\u2010MALS\u2010FLD workflow was optimised to enable the analysis of EV surface markers CD9, CD63 and CD81, and cancer biomarkers PSMA, EpCAM and HER2 in samples of increasing complexity, including purified EV preparations, cell culture supernatant, urine and blood plasma. 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