A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.