BACKGROUND: Oxidative stress, a source of genotoxic damage, is often the underlying mechanism in many functional cell disorders. Extracellular vesicles (EVs) have been shown to be key regulators of cellular processes and may be involved in maintaining cellular redox balance. Herein, we aimed to develop a method to assess the effects of EVs on DNA oxidation using porcine seminal plasma extracellular vesicles (sEVs). RESULTS: The technique was set using a cell-free plasmid DNA to avoid the bias generated by the uptake of sEVs by sperm cells, employing increasing concentrations of hydrogen peroxide (H CONCLUSIONS: Our results suggest that the surface of small sEVs, including the peripheral corona layer, may exert a protective function against alterations that are originated by oxidative mechanisms. In addition, our in vitro study opens path to detect, localize and quantify the protective effects against oxidation of extracellular vesicles derived from different fluids, elucidating their function in physiopathological states.