Characterization of post-translational modifications in proteins is one of the major tasks that is to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification that regulates enzymatic activity, subcellular localization, complex formation and degradation of proteins. Rapid advances in mass spectrometry instrumentation coupled to the development of analytical methods over the past several years now allow us to investigate the phosphoproteome on a global scale.