Extracellular vesicles (EVs) play a crucial role in intercellular communication. Characterizing EV protein composition is essential to understand EV function(s). Isolating EVs from cell culture medium is a common approach to study EVs, but it remains unclear whether EVs isolated from in vitro conditions accurately reflect physiological conditions of the same source in vivo tissues. Here, we analyzed the protein composition of EVs isolated from freshly dissected mouse forebrain and primary dissociated mouse forebrain culture medium. In total, 3,204 and 3,583 proteins were identified in EVs isolated in vivo and in vitro, respectively. Among the proteins identified from both EV sources, there was substantial overlap (~86%). While the overall proteome compositions were very similar, in vitro EVs were relatively enriched with transmembrane/GPI-anchored membrane and cytosolic proteins (MISEV2023 category 1 and 2) typically associated with EVs. Conversely, while both in vivo and in vitro EVs express likely non-EV proteins (MISEV2023 category 3), the in vivo samples were significantly more enriched with these probable contaminants, specifically ribosomal proteins. Our findings highlight that in vitro EVs may be representative of in vivo EVs when isolated from the same source tissue using similar methodology; however, each population of EVs have differences in both total and, primarily, relative protein expression likely due to differing levels of co-eluting contaminants. Therefore, these points must be considered when interpreting results of EV studies further suggesting that improved methods of isolation to reduce non-EV contaminants should be further investigated.