This study aims to investigate the small extracellular vesicle (sEV) impact on the blood-brain barrier (BBB), particularly the interactions between sEVs and brain endothelial cells (BECs). The molecular components governing these interactions are currently unknown. In order to identify sEV-BEC interactors, a novel assay based on proximity-dependent biotinylation was developed. sEVs purified from a panel of cell lines derived from various peripheral and brain-derived tumors, including HEK293 embryonic kidney cells, SK-MEL-28 skin melanoma, A549 lung adenocarcinoma, MDA-MB-231 breast adenocarcinoma, 1321N1 astrocytoma, SH-SY5Y neuroblastoma, MO3.13 oligodendocytes, and HMC3 microglia, were labelled with Cell Tracker™Orange. These labelled sEVs were then applied to the immortalised brain endothelial cell line hCMEC/D3 in an optimised Transwell® setup to assess BBB barrier integrity and sEV uptake. The interactions between sEVs and BECs exhibited cell origin and donor compartment-dependent effects on barrier tightness and sEV internalisation. For instance, SH-SY5Y sEVs showed 6 times greater uptake than SK-MEL-28 sEVs. Comparative proteomics revealed cell-specific sEV proteomes, including 458 SH-SY5Y sEV-specific proteins. sEVs from HEK293 and SH-SY5Y cells expressing a GPI-anchored biotin ligase (BioID2-GPI) were purified (BioID-GPI-sEVs) and applied to BECs. Biotinylated proteins (potential interactors) were isolated by Streptavidin pull-down and identified by mass spectrometry and will be investigated in more detail. Overall, applying BioID2-GPI-sEVs on BECs is a promising new approach to discover sEV-BBB interactors, enabling the tracing of sEV pathways in BECs. This understanding of sEV cellular fate at the BBB could open avenues for innovative drug delivery methods in brain disease treatment.