MALDI in-source decay (ISD) was first introduced in 1995 in a landmark paper by RS Brown and JJ Lenon (Anal. Chem. 1995, 67, 3990–3999). Although MALDI MS is considered a soft ionization technique which enables the analysis of high molecular weight proteins exceeding several hundred kDa in an intact manner, it became apparent that some analyte fragmentation occurs on a very short time scale during the desorption ionization process. ISD is the combination of laser induced fragmentation and rapidly occurring metastable decay. This phenomenon can be used to obtain primary sequence information on peptides and (small) proteins also referred as top down sequencing. Beyond its analytical utility, the ISD fragmentation mechanism is currently being studied by several groups. In this perspective special feature article, the MALDI-ISD mechanism is reviewed and a specific fragmentation mechanism is introduced and studied in details: the N-terminal side of Cys residue is described to promote the generation of c' and w ISD fragments. Dr Edwin De Pauw is Professor of Chemistry at Liege University in Belgium. One of the objectives of his mass spectrometry laboratory is to contribute to the development of new fundamental concepts and to enlighten their applications in the fields of analytical chemistry.