Ladder sequencing of polypeptides involves progressive N- or C-terminal amino acid truncation via chemical or enzymatic treatments. Peptide ladders are generated in which each component differs from the next by one residue. The ladder components are analyzed by mass spectrometry, and the amino acid sequence is deduced from the mass differences between consecutive fragments. Chemical procedures are common in N-terminal degradation, whereas proteolytic digestion is often used in C-terminal sequence analysis. Matrix-assisted laser desorption/ionization mass spectrometry is widespread for one-step readout of the peptide ladders and provides high sensitivity in combination with robustness and ease of use. The particular advantage of ladder sequencing in relation to other techniques for sequence analysis is the high data acquisition rate and the very good sample throughput that can be achieved. Multiple determinations are carried out within minutes at high sensitivity and low sample consumption. Several reports demonstrate analysis at the low picomole to femtomole level.