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Abstract Background Centrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co‐isolation with extracellular vesicles (EVs). Objective To assess the ability of rate zonal centrifugation (RZC) to separate platelets from co‐isolated EVs. Methods Using a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet‐derived EVs (n = 3). After RZC, all fractions were collected and stained with anti‐CD61‐Alexa 488 to measure the concentrations of platelets and platelet‐derived EVs by flow cytometry. Results We confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8‐19, whereas the majority of platelet‐derived EVs are detectable in fractions 1‐7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based. Conclusion RZC can partially separate platelets from EVs.