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Recently, extracellular vesicles (EVs) have been developed as therapeutic targets for various diseases. Biodistribution is crucial for EVs intended for therapeutic purposes because it can determine the degree of on- and off-target effects. This study aimed to explore techniques to evaluate the biodistribution of unmodified EVs. We devised a novel quantitative polymerase chain reaction (qPCR)-based assay to detect unmodified EVs by targeting mitochondrial deoxyribonucleic acid (mtDNA), a constituent of EVs. We focused on specific mtDNA regions that exhibited homologous variations distinct from their rodent mtDNA counterparts to establish this analytical approach. Herein, we successfully designed primers and probes targeting human and rodent mtDNA sequences and developed a highly specific and sensitive qPCR method. Furthermore, the quantification range of EVs isolated from various cells differed based on the manufacturer and cell source. IRDye 800CW-labelled Expi293F EV mimetics were administered to the animals via the tail vein to compare the imaging test and mtDNA-qPCR results. The results obtained from imaging tests and mtDNA-qPCR to investigate EV biodistribution patterns revealed differences. The results revealed that our newly developed method effectively determined the biodistribution of unmodified EVs with high sensitivity and reproducibility., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)