Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), a well-suited method for the characterization of peptides and proteins, was used for analysis of protected peptide fragments. It is shown that acidic matrices, e.g. 2,5-dihydroxybenzoic acid, frequently used in MALDI-TOF MS of peptides, causes partial cleavage of acid-labile side-chain protecting groups. Because this effect is strongly related to the matrix used, the observed deprotection can be avoided by choosing an appropriate matrix such as 2,4,6-trihydroxyacetophenone or 2-amino-5-nitropyridine. The advantage of neutral matrix compounds for MALDI-TOF analysis of protected peptides is clearly demonstrated, confirming the potential of MALDI-TOF mass spectrometry.