The inaccessibility of the brain and spinal cord makes studying biochemical changes in the central nervous system (CNS) difficult. Traditional methods include brain imaging and biochemical analysis of the cerebrospinal fluid, which are not ideal. CNS-originating extracellular vesicles (EVs) isolated from blood have emerged as a promising source of biomarkers for a variety of CNS studies, because they can cross the blood-brain barrier and protect their cargo, including proteins, nucleic acids, carbohydrates, and lipids, providing a glimpse into the biochemical status of the originating cells. Importantly, immunoprecipitation using antibodies against cell-specific EV surface markers allows isolating CNS-originating EVs from blood and other easily accessible biofluids. High-sensitivity methods can then analyze the EV contents to measure candidate biomarkers or be used for biomarker discovery. However, in some downstream applications, such as mass-spectrometry-based proteomics or surface-enhanced Raman spectroscopy, nonspecific binding of high-abundance biomolecules in the biofluid may prevent using CNS-originating EVs. In these cases, EVs isolated from the culture medium of neurons or glia may provide an alternative, because an immunoprecipitation step is unnecessary. Here, we briefly discuss the strategy of analyzing biomarkers in such EVs and provide detailed protocols for isolating CNS-originating EVs from blood products and neuronal-culture media.