Extracellular Vesicles (EVs) transfer molecules from donor to acceptor cells. The EV-content delivery process within the acceptor cell is still unknown. We developed a protease protection-based assay to assess EV-content release in vitro. We found that EV-cytosolic cargo is released from EVs when isolated vesicles are incubated with purified plasma membrane sheets at acidic pH, a signature of endosomal environment. This process is protein-dependent. Our results suggest that EV-content delivery occurs within the endosomes of the acceptor cell and is triggered by acidification. This   process might involve membrane fusion.