Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.