Extracellular vesicles (EVs) are small lipid vesicles secreted by cells that originate from the cell surface (typically enriched in CD9) or from multivesicular bodies (typically enriched in CD63). While current methods for studying EVs involve concentrating and purifying EVs, they do not provide information about the distance or amount of EVs that may transfer from one cell to another. Here, we adapted a coculture assay previously developed (Burtey et al. FASEB 2015) in which MCF-7 cells - a model for human mammary epithelial cells – were color-coded by expressing EV-markers hCD63 and/or hCD9 or hCD81 fused with fluorescent mCherry or GFP with untransfected “acceptor” cells labelled with Cell-Tracker-Blue, a non-transferrable dye. By 3D quantitate high-resolution confocal fluorescence microscopy, we observed the direct transfer EV markers between cells, quantified the distance and the rate of transfer for the different EV markers. Results showed that CD81 did not transfer far away from the donor cells while CD9 reached more distant acceptor cells. Similar results were obtained for endogenous CD81, CD9 and CD63 whose transfer were analyzed by immunofluorescence on cocultures of wt MCF-7 cells to CRISPR KO CD63, CD81 or CD9 MCF-7 cells. Simultaneously monitoring of endogenous CD9 and CD81 transfer was done by coculturing wt cells with double KO CD9/CD81 cells and revealed that EV markers colocalized in acceptor cells suggestive of their cotransfer. Interestingly, 3D analysis indicated the abundance of punctate structure containing CD81 and CD9, or CD63 at the basal level, below acceptor cells or on the substrate. Altogether, these results suggest that this coculture approach is highly suitable for direct qualitative and quantitative imaging of EV-transfer, in living or fixed cells, and is also suited to screen for EVs cargoes or regulators and further decipher the mechanisms underlying EV transfer.