This study presents a comparative study of different methodologies for the extraction of proteins from platelet derived-extracellular vesicles (pEV) prior to subsequent mass spectrometry (MS) analysis. Protein isolation is performed through three different methodologies based on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), organic solvent precipitation (OSP) or magnetic beads (MB), followed by protein digestion and sample acquisition by LC-MS/MS.