Synthetic enzyme substrates were first used to identify lac mutants in Escherichia coli in the late 1940s, but this technology was not used in food microbiology until the late 1980s. Owing to the increased specificity afforded by chromogens and fluorogens in the detection of pathogens, their use has steadily increased. A major obstacle to greater understanding and use of chromogenic and fluorogenic substrates by food microbiologists is a lack of understanding of chemical nomenclature and molecular biology. Details of the relationship between natural sugars and synthetic analogues are given, together with a description of the molecular biology involved in the induction of operons, transportation and hydrolysis of substrates. The nature of a variety of fluorogens, chromogens and Trojan horses is discussed, together with the benefits this technology can bring in food analysis.