Pepscan is a procedure for mapping and characterizing epitopes involving the synthesis of overlapping peptides and analysis of the peptides in enzyme-linked immunosorbent assays (ELISAs). The method is based on combinatorial chemistry and was pioneered by Mario Geysen and coworkers. Twenty-five years later, the Pepscan methodology, evolved and modernized with the latest insights, is still an important part of Pepscan’s epitope mapping platform, which is instrumental in therapeutic antibody development.