Clostridioides difficile is an antibiotic resistant bacterial human pathogen that colonises the large intestine in the absence of a healthy competing gut microbiota. Current treatments for C. difficile infection (CDI), the most common cause of healthcare-associated gastrointestinal disease, entail use of antibiotics that perpetuate gut dysbiosis and enable disease recurrence. This highlights an urgent requirement to further understand the fundamental biology of this pathogen so novel therapies can be sought. A promising, albeit unexploited, target for new therapeutics is the process by which C. difficile forms spores, dormant cell forms resistant to environmental stressors and the primary CDI infectious agent. Sporulation begins with an asymmetrical cell division, and entails substantial peptidoglycan remodelling as the smaller forespore is engulfed by the larger mother cell prior to spore maturation. The molecular details of sporulation require further unravelling to find potential pharmaceutical targets to interrupt this process, thereby eliminating the CDI transmission route. SpoIIP is a dual amidase and endopeptidase involved in peptidoglycan remodelling during engulfment, but the localisation of SpoIIP in vivo and the identity of its endopeptidase catalytic residues remain unknown. Previous work has identified two cleavage sites targeted by the SpoIVB2 peptidase, proposed to be a mechanism of liberating enzymatically active SpoIIP isoforms. Our current work seeks to further investigate this, with each isoform having been generated using inverse PCR and tested for enzymatic activity using peptidoglycan digestion assays. By furthering understanding of the mechanisms of forespore engulfment, the crucial pursuit of novel CDI therapies will be aided.