Updated project metadata. The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probed the effect of starting volume for the EV proteomics profile. Using proteomics, SEC fractions 3-5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4-5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6ml, 3ml, 1ml, and 0.5ml) to evaluate the effect on the proteomic profile. A total of 180 proteins was shared regardless of the starting volume. With a 0.5ml starting volume, 743 ± 77 or 345 ± 88 proteins were identified depending on the MaxQuant settings (with and without ‘match between runs’ active). The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5ml of canine CSF.