Proteomics offers the opportunity to comprehensively investigate the anucleate platelet. Here, we present a detailed procedure for enrichment by immunoprecipitation, using the monoclonal antibody 4G10, of the dynamic phosphotyrosine proteome of human platelets. Such an approach offers the possibility of capturing the dynamic tyrosine phosphorylation events that occur upon platelet activation and aggregation, with an aim to identify novel signaling proteins.