In an approach to gain insight into the sequence-dependent dephosphorylation of multiple phosphotyrosyl-containing peptides by the phosphatases SHP-1 and PTP1B, we applied a chromatographic technique for the analysis of the dephosphorylation products. Mono-, bi- and triphosphorylated reference peptides corresponding to positions 1999-2014 in the activation loop of the receptor tyrosine kinase Ros were first analyzed by reversed-phase HPLC and MALDI-TOF/TOF mass spectrometry. Then, the respective products from enzymatic treatment were investigated by HPLC and compared to the standard peptides. The results obtained in this study emphasize the advantage of monitoring phosphatase reactions for mono- and biphosphorylated peptides using the described procedure rather than spectrophotometric and fluorimetric methods that do not allow for a clear identification of the products formed.