This unit describes a procedure for proteolytic digestion of a (32)P-labeled protein, followed by separation of the digestion products in two dimensions on a TLC plate, which gives rise to a phosphopeptide map. Phosphopeptides present on the TLC plate are visualized by autoradiography. These maps give information about the number of phosphate-containing peptides in the digest, and this is related to the number of phosphorylation sites present in the protein. Phosphopeptide maps can also be used to find out whether the sites of phosphorylation on a protein change upon treatment of cells with certain agents. Procedures are also provided for the identification of the sites of phosphorylation from the phosphopeptide maps. A support protocol details purification of phosphopeptides by HPLC and subsequent analysis by mass spectrometry or peptide microsequencing.