This study characterizes the EV-derived proteome secreted by three major cell types of the neurovascular unit: human brain endothelial cells (HBECs), astrocytes, and neurons. Mass spectrometry analyses of EVs and corresponding cells were performed to assess the functional roles of EVs in brain intercellular communication. This study characterizes the EV-derived proteome secreted by three major cell types of the neurovascular unit: human brain endothelial cells (HBECs), astrocytes, and neurons. Mass spectrometry analyses of EVs and corresponding cells were performed to assess the functional roles of EVs in brain intercellular communication.