Different search programs were compared to judge their particular efficiency in protein identification. We established a human blood platelet protein map and identified tyrosine‐phosphorylated proteins. The cytosolic fraction of human blood platelets was separated by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) and phosphorylated proteins were detected by Western blotting using anti‐phosphotyrosine antibodies. Visualized protein spots were excisted, digested with trypsin and analyzed by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). The obtained mass fingerprint data sets have been analyzed using ProFound, MS‐Fit and Mascot. For those protein spots with no significant search results MALDI post source decay (PSD) spectra have been acquired on the same sample. For automatic interpretation of these fragment ion spectra, the SEQUEST and Mascot algorithm were applied. Another approach for the identification of phosphorylated proteins is immunoprecipitation using an anti‐phosphotyrosine antibody. A method for immunoprecipitation of tyrosine‐phosphorylated peptides was optimized.