Synthetic peptides have been shown to be valuable tools for viral laboratory diagnosis and can provide uniform, chemically well-defined antigens for antibody analysis, reducing inter- and intra-assay variation. The main aim in the development of peptide-based diagnostic tests is to recognise specific antibodies induced by the whole viral proteins but using selected short fragments containing the most potent antigenic determinants. The success of this approach depends on the extent to which synthetic peptides are able to mimic the immunodominant epitopes of antigens. In recent years, synthetic peptides that mimic specific epitopes of infectious agents proteins have been used in diagnostic systems for various human diseases. The present review summarizes some of the drawbacks of the use of relatively short linear peptides as antigenic substrates and the subsequent chemical strategies developed in order to overcome the low peptide reactivity against specific antibodies. Moreover, it outlines the most significant bibliography published in the last five years which provides validated peptide based tests potentially useful for diagnosis of viral, bacterial, parasitic and autoimmune diseases.