Platelets (thrombocytes) are the smallest human blood cells and are pivotal in processes of hemostasis and thrombosis. Central to their function, the activation of platelets includes a complex interplay of adhesion and signalling molecules mediated via the plasma and inner membrane. Because platelets are enucleated, the analysis of the proteome is the best way to approach their biology. Here, we employ mass spectrometry (MS)-based proteomics to characterise membrane proteins derived from non-stimulated human platelets. This protocol details the extraction and purification of platelet membrane proteins from whole blood using SDS-PAGE in conjunction with LC-MS/MS. This method allowed the identification, and characterization of 207 platelet membrane proteins (PMP) from approximately 9.95 x 10(9) platelets (16).