Extracellular vesicles (EVs) are mediators of intercellular communication in diverse cellular functions. Visualizing EVs in vivo is important to elucidate the biogenesis of EVs, and various approaches have been developed for in vivo EV tracking. The ubiquitously expressed tetraspanin CD63 is liberally incorporated into EVs. Thus, fluorescently tagged CD63 has been used in many studies to label EVs. In the present study, we presented isolation and transfer assays for EVs from two transgenic rats expressing CD63-GFP in their body fluids or brains.