Cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) are significant media for the cell-cell interaction in the central nervous system (CNS). Thus, characterization and analysis of CSF EVs can provide a pile of information for the discovery of molecular biomarkers of CNS diseases. Furthermore, phosphorylation of proteins plays a vital role in the regulation of protein function and cell signal transduction mechanism, and dysfunction of phosphorylation is always associated with the occurrence and progression of disease. Aiming to conduct mass spectrometry (MS)-based phosphoproteomic analysis of CSF EVs, we describe here a complete workflow to isolate EVs from CSF and profile their phosphoproteome, employing EVtrap-based EV isolation, spin-tip PolyMAC-Ti phosphopeptide enrichment and label-free quantitation of phosphoproteome. The approach provides a powerful CSF EV phosphoproteome analysis pipeline that can contribute to the discovery and identification of EV-based molecular markers.