Extracellular vesicles (EVs) are implicated in the pathogenesis of neurodegenerative diseases and offer a source of biomarkers for diagnostic, stratification and therapeutic monitoring purposes. A major limitation in the development of specific biomarkers based on cerebrospinal fluid (CSF) EVs is the difficulty of extracting sufficient quantities of vesicles for discovery techniques such as proteomics. This study describes a novel application of a size-based extraction technique for CSF EVs using ultrafiltration liquid chromatography (UFLC), and comparison with the gold-standard ultracentrifugation (UC) method.