Extracellular vesicles (EVs) are promising biomarkers, yet their proteomic analysis from plasma is hampered by low abundance and co-purification of contaminants (e.g., lipoproteins, platelets) and technical variability, particularly in small-volume animal models. We developed and validated a modular protocol integrating Size Exclusion Chromatography (SEC) with Strong Anion Exchange (SEC-SAX), specifically tailored for quantitative LC-MS proteomics from small starting volumes (150 μl) of plasma. SEC alone successfully removed 99% of Albumin, and the SAX step significantly enriched EVs over contaminating lipoproteins. Downstream single pot solid phase enhanced (SP3) sample prep and STAGE tip solid phase extraction ensured maximum proteome depth. Critical confounding factors were objectively assessed: Platelet Factor 4 (PF4) was confirmed as a highly sensitive platelet marker, confirming the necessity of meticulous plasma preparation. Sample haemolysis impacted the plasma EV proteome data. As such, an objective measure (nanodrop spectrophotometer) of haemolysis and exclusion of haemolysed samples (heme >0.3 mg/ml) is recommended. The protocol is applicable to both human and mouse plasma. Quantitative analysis of EVs from eight individual mouse samples demonstrated robust EV enrichment and enabled detection of biomarker proteins relevant to neurodegenerative diseases.