Since the last decade, the field of extracellular vesicles (EVs) has increasingly grown. These “intracellular messengers” became interesting as biomarker source in the SNC research, because of their content, which may reflect the physiological state of their donor cells in a context of an external stimuli. However, still little is known about EVs released from cell types constituting the blood-brain barrier (BBB), especially on the human brain microvascular endothelial cells (HBMECs). The current study aimed to isolate and characterize EVs from HBMECs and to analyze the EVs proteome modulation after an external stimulate such as the Paraquat (PQ), a neurotoxicant. Size distribution, quantity and presence of EV markers were assessed. Identification and quantification of EVs proteins was conducted by data-independent acquisition mass-spectrometry (DIA-MS). Signature pathway of PQ-treated EVs were analyzed by gene ontology terms and pathway enrichment. Results present evidence that PQ-treated HBMECs and EVs are sharing proteins belonging to the ubiquinone metabolism and to the transcription HIF-1 targets pathways with a highly significant p-values, suggesting that those pathways may be a potential EVs signature of the PQ-induced toxicity in the BBB.