Proteolytic enzymes (proteases) are integral to multiple physiological processes, and increased protease activity is observed in inflammatory bowel disease (IBD) and other gastrointestinal diseases. Therefore, protease activity has been explored for potential diagnostic biomarkers and therapeutic targets. Faecal samples are commonly used to measure protease activities associated with gastrointestinal conditions as they allow non-invasive access to the diseased environment. However, faecal sample storage and protein extraction methods are not standardised for the measurement of faecal protease (FP) activity. In addition to being of scientific importance, the evaluation of the viability of FP activity as a potential biomarker for IBD management is also of clinical interest. In this study, the effect of different sample storage conditions and extraction techniques on FP activity were measured, on samples from IBD patients, using fluorescent substrates highly selective for serine proteases. FP activity was found to be stable for up to 72 hours at both 4 degrees C and room temperature. Activity was found to decline more rapidly when samples were stored in extraction buffer rather than as crude samples. Regarding extraction techniques, FP activity was highest when extracted in Tris based buffers at pH 7.5-8; bead-beating of samples during extraction was found to have no significant effect on activity; and processing the extracted sample on ice was shown to minimise loss of FP activity. These results provide a framework for standardised evaluation of FP activity and suggest that protease activities have the potential to be a viable future biomarker.