Clostridioides difficile, a spore forming bacterium, causes antibiotic associated diarrhoea and is a major public health concern. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Recently, our team identified that the sporulation specific penicillin binding protein, CdSpoVD is essential for spore formation in C. difficile. Interestingly, CdSpoVD was inactivated by the cephamycin class of β-lactam type antibiotic, and treatment of C. difficile with cephamycins was able to inhibit sporulation. Co-treatment with the current standard of care, Vancomycin, and the cephamycin Cefotetan, was able to prevent diseases relapse in a mouse model of C. difficile infection, suggesting that CdSpoVD would be an attractive molecular target for anti-sporulation agents. This undergraduate research project aimed to identify the active site of CdSpoVD to facilitate future computational modelling and drug design. The penicillin binding proteins have a well-characterised mechanism of action where an active site serine residue forms a covalent bond with the β-lactam ring of the antibiotic. Bioinformatic analysis of CdSpoVD suggested two possible serine residues (S276 and S332) that may act as the active site serine. In this project, we produced single point mutations to individually change each serine to alanine. Mutant CdSpoVD proteins were expressed, purified and assessed for their ability to bind a fluorescently labelled b-lactam type antibiotic. The results show that S276 is the active site serine residue, however, it appears that S332 likely has a role in the active site, either in substrate positioning or active site architecture. This data will allow in silico molecular docking of the cephamycins to produce structure-activity relationships for future rational design of novel anti-sporulation agents.