<p>Alzheimer’s disease (AD) is characterized by the formation of senile plaques and neurofibrillary tangles, mainly composed of amyloid-β (Aβ) peptide aggregates and hyperphosphorylated tau protein, respectively. AD pathophysiology is highly complex, involving multiple abnormal cellular pathways linked to disease progression. Recently, extracellular vesicles (EVs) have emerged as potential contributors to disease development. Thus, this study explored the proteome of neuronal EVs under conditions that mimic Alzheimer’s disease by employing mass spectrometry in EVs isolated from N2a cells treated with Aβ. Bioinformatic analysis revealed proteins involved in signal transduction, post-translational protein modification, translation, and proteolysis. Furthermore, Aβ treatment led to either an enrichment or scarcity of proteins related to cytoskeletal and mitochondrial dynamics, calcium-dependent signalling, phosphorylation, as well as proteins involved in Aβ production and aggregation. Overlap between EVs’ proteome&#xa0;upon Aβ treatment and key AD-related proteins identified glycogen synthase kinase 3β (GSK3β) as a central node in the resulting protein interaction network. Additionally, the GSK3β interactome, derived from the EVs’ proteome, highlighted protein phosphatases as relevant EVs’ cargo under Alzheimer’s disease mimicking conditions. The activity of GSK3β and protein phosphatases in EVs was monitored, revealing significant differences between control and Aβ-treated conditions. These findings support not only that EVs carry key proteins involved in phosphorylation dynamics but also that Aβ treatment alters EVs’ proteomic profile, potentially impacting AD development. Proteomic changes in EVs may provide valuable insights into the mechanisms underlying AD and also contribute to the identification of novel potential therapeutic targets.</p>