Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) is a powerful tool to separate complex protein mixtures including whole cell lysates. In combination with immunoblotting techniques or radioactive labeling techniques it is a fast and convenient way to demonstrate the presence of certain proteins or protein modifications. With the development of extremely sensitive analytical techniques such as matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) or electrospray ionization (ESI)‐MS, it has become possible to use 2‐D gels not only as an analytical but also as a preparative tool. Starting with a number of spots excised from 2‐D gels, a protein can be identified using different strategies involving enzymatic cleavage of the protein in the gel matrix, elution of the resulting peptides and analysis of these peptides by mass spectrometry. The obtained peptide mass fingerprint or fragment ion spectra from peptides can be used to screen protein or nucleic acid databases in order to identify the protein. We have used the techniques described above to identify proteins from human platelets which change their phosphorylation state following activation of platelets by thrombin. Platelets were radioactively labeled with [³²P]orthophosphate and stimulated. Several protein spots in the observed range of 10–80 kDa and an isoelectric point of 3–10 showed a significant increase or decrease in phosphorylation. We present the results from the investigation of a spot group representing different isoforms and phosphorylation states of myosin light chain.