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.