Blood platelets are important components of haemostasis. After their activation they cause healing of wounds by forming plugs and initiate repair processes. One important event in regulating this activation is the phosphorylation/dephosphorylation of multiple proteins on various tyrosine, serine and threonine residues. To understand the exact molecular mechanisms in platelet activation it is essential to identify proteins involved in the signalling pathways and to localise and characterise their phosphorylation sites. After treatment with ³²P and separation by 2D-PAGE using different pI ranges, phosphorylated platelet proteins were detected by autoradiography. Phosphotyrosine-containing proteins were assigned by immunoblotting with an anti-phosphotyrosine antibody. Another approach for the identification of phosphorylated proteins was immunoprecipitation of tyrosine-phosphorylated proteins using an anti-phosphotyrosine antibody. Protein spots/bands of interest were excised from the gel, digested with trypsin and analysed by MALDI-TOF-MS and nano-LC-ESI-MS/MS, respectively. Several phosphorylated proteins could be identified and the localisation of some in vivo phosphorylation sites was possible.