Dipeptidyl peptidase-4 inhibitors (DPP4i) are a class of orally available, small molecule inhibitors for the management of Type-II diabetes. A rapid, real-time, functional breath test for DPP4 enzyme activity could help to define DPP4i efficacy in patients that are refractory to treatment. We aimed to develop a selective, non-invasive, stable-isotope 13 C-breath test for DPP4. In vitro experiments were performed using high (Caco-2) and low (HeLa) DPP4 expressing cells. DPP gene expression was determined in cell lines by qRT-PCR. A DPP4 selective 13 C-tripeptide was added to cells in the presence and absence of the DPP4 inhibitor Sitagliptin. Gas samples were collected from the cell headspace and 13 CO 2 content quantified by isotope ratio mass spectrometry (IRMS). DPP4 was highly expressed in Caco-2 cells compared to HeLa cells and using the 13 C-tripeptide, we detected a high 13 CO 2 signal from Caco2 cells. Addition of Sitaglitpin to Caco2 cells significantly inhibited this 13 CO 2 signal. 13 C-assay DPP4 activity correlated positively with the enzyme activity detected using a colorimetric substrate. We have developed a selective, non-invasive, 13 C-assay for DPP4 that could have broad translational applications in diabetes and gastrointestinal disease.