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Their capacity to influence complex intracellular pathways highlights their therapeutic potential in addressing various conditions, including neurodegenerative diseases and cancer. A novel capillary isotachophoresis (cITP) method was developed for the electrokinetic characterization of pre\u2010isolated EVs. Distinct peaks could be resolved at near\u2010baseline resolution using a novel mixture of spacer ions and laser\u2010induced fluorescence (LIF) detection. The vesicles were effectively separated from the unbound carboxyfluorescein diacetate succinimidyl ester (CFDA\u2010SE) amine\u2010reactive fluorescent stain used to detect them and from residual contaminants. The identity of the peaks shown in the electropherograms was validated via various methods, including incubation with specific antibodies or spiking of putative contaminants, such as proteins and lipoproteins. 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