The analysis of protein phosphorylation sites is one of the major challenges in the post-genomic age. To understand the role of reversible phosphorylation in cell signaling, the precise location of phosphorylation sites must be determined in a phosphoprotein as well as the effect that these post-translational modifications have on the function of the protein. The use of solid phase Edman degradation of (32)P-labeled phosphoproteins and peptides was described over 10 years ago as a method for the identification of phosphorylation sites. Since that time a number of laboratories have used this technique as the standard method for phosphorylation site analysis. In this report, we will describe how we routinely use this technology to perform hundreds of successful phosphorylation site analyses per annum. By combining mass spectrometry to identify the phosphopeptide and solid phase Edman degradation to localize the site of phosphorylation, subpmole quantities of phosphopeptides can be successfully characterized.