As a chemical reaction takes place, it passes through a high energy, unstable structure called the transition state which is positioned between the chemical structures of reactants and products. Enzymes function by lowering the energy barrier to form the transition state, thereby increasing the rate of reaction. Knowledge of the transition state is critical to understanding enzyme mechanisms and importantly, can be exploited to generate new drugs. Drugs that mimic the transition state structure of an enzyme reaction are known as transition state analogues and are powerful enzyme inhibitors. My research program at the University of Sydney involves using transition state analysis to solve the transition state structures of glycosyltransferases. Glycosyltransferases catalyse the formation of glycosidic bonds and do this by transfer of a sugar from a nucleoside diphosphate to an acceptor molecule that includes proteins sugars and lipids. I will present an overview on the development of transition state analogues that inhibit TcdA and TcdB from Clostridioides difficile. C. difficile is an opportunistic pathogen that colonises the large intestine and causes a range of gastrointestinal diseases including life-threatening diarrhea. C. difficile pathology is mediated by TcdA and TcdB which contain a glycosyltransferase domain that glycosylates host Rho GTPases. Glycosylation of Rho GTPases leads to changes in the actin cytoskeleton which leads to a loss of intestinal integrity. Transition state analogues, isofagomine and noeuromycin inhibit TcdA and TcdB glycosylation of Rho GTPases and importantly, prevent C. difficile induced mortality in mouse model of C. difficile infection.