Research on the pleuromutilin class of antibiotics has spanned 60 years with many analogues prepared and published in the patent and scientific literature. The seven analogues in the Pleuromutilin Analogue Set represent pivotal advances in the development of the pleuromutilin class, with the analogues displaying a range of physico-chemical, potency, spectrum of action and cross-resistance profiles. The Pleuromutilin Analogue Set is a useful tool in addressing current research questions on antibiotics using the resources accumulated since the discovery of pleuromutilin in 1951.