Platelets exert a crucial function in haemostasis, wound repair, and the formation of vascular plugs, underlying thrombotic diseases such as stroke and myocardial infarction. Analysis of platelet biochemistry is largely dependent on protein analysis as platelets are anucleated cells providing little analytical target for DNA or RNA based strategies. Here we present data from our analysis of the human platelet proteome, the entire set of proteins building a platelet at a given point in time. Proteins were separated by two‐dimensional electrophoresis (2‐DE) using broad and narrow range pH gradients in the isoelectric focusing step. Consequently, a high‐resolution 2‐DE proteome map has been generated that comprises approximately 2300 different protein features. From the 536 protein features detected in the 4–5 pI range 284 features were identified by electrospray ionisation time of flight tandem mass spectrometry. These 284 proteins originate from 123 different open reading frames. This includes the five human proteins KIAA0193, KIAA0573, KIAA0830, WUGSC:H_DJ0777O23 protein, and cytokine receptor related protein 4, all isolated for the first time. The data are discussed with regard to proteome characteristics, protein function, and the high prevalence of signalling molecules. This study contributes to a more thorough and holistic understanding of platelet biology, helping to build the basis for future identification of new drug targets and therapeutic strategies.