We have developed a new method for the quantification of phosphoserine, phosphothreonine, and phosphotyrosine as dabsyl derivatives in acid-hydrolyzed extracts of 32P-labeled A431 cells. In the first step the phosphamino acids are concentrated using a disposable anion-exchange column. Subsequently, the phosphoamino acid fraction is treated with dabsyl reagent (28.8 mM) for 10 min at 70 degrees C. After cleanup with a second anion-exchange column followed by separation on a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC sheets using a one-dimensional solvent system. The major advantages of this method are the complete separation of dabsylated P-Ser, P-Thr, and P-Tyr on silica aluminum sheets in a very reproducible way without the interference of 32P contaminants originating from hydrolyzed cell extracts. Very clean chromatograms are obtained, enabling the fast and unambiguous quantification of the phosphoamino acids by simply cutting out the relevant spots from the aluminum sheets. A high sensitivity is achieved by the removal of the amino acids before derivatization of the sample. This allows the use of relatively low amounts of [32P]orthophosphate to load up the cells. Most important, the method allows the simultaneous analysis of dozens of samples within 1 day, making it a very convenient technique for routine analysis of the phosphorylation state of cultured cells. Consequently the method is well suited to implementation in large screenings for inhibitors of protein kinases, e.g., PTK inhibitors, in whole-cell studies.