We have developed a new workflow to unambiguously localize phosphorylation sites on proteins. We demonstrate that spectral matching of phosphopeptide datasets against a library of the well-simulated spectra provided higher sensitivity for confident site localization than other tested programs. To computationally simulate tandem mass spectra representing all possible singly phosphorylated forms of a peptide, characteristic fragment ions are predicted from ions of their dephosphorylated form generated by beam-type collision-induced dissociation.