A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using ATP-g-S followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity at low pH of the sulfur in thiophosphate residues, whereas the nucleophilicities of phosphates, amines and other functionality of amino acids are negligible or significantly suppressed. Modified alkylation reagents linked to biotin or solid supports (e.g., glass or sepharose beads) with or without a photocleavable linker facilitate the isolation of the thiophosphorylated peptide/proteins. This approach is demonstrated through the localization of phosphorylation sites on myosin regulatory light chain. We anticipate this technique will be useful for isolation and subsequently identification of newly thiophosphorylated proteins, produced either in vivo or in vitro, thus facilitating the dissection of protein phosphorylation networks.