ABSTRACTTherapeutic peptides targeted at various diseases are becoming increasingly relevant for the pharmaceutical industry. Several of these drugs were originally designed by mimicking a segment of a protein of interest. As such, protein mimicry represents a promising strategy both in immunology, for the identification of B‐ and T‐cell epitopes, as well as for the modulation of protein activity, including the disruption of protein–protein interactions (PPIs) and the interference with biological or pathological cellular functions. Several methods have been developed to pinpoint the (binding) epitopes of a protein or the regions responsible for biological activity. One of such strategies is the scanning of the protein or selected domains with synthetic overlapping peptides. As the mechanism of action of a mimetic peptide can be similar to that of the whole protein, this method offers a powerful tool for the investigation of protein function, along with providing a solid basis for the development of therapeutic candidates. This review gives a general overview of different applications of the peptide scanning methodology, describing a comparison of the preparation and use of solid‐phase libraries (peptide arrays) with isolated peptide libraries and highlighting their strengths and most common applications.