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Abstract Cytochrome P450 (CYP) 3A4 plays a major role in drug metabolism. Its activity could be determined by non-invasive and cost-effective assays, such as breath analysis, for the personalised monitoring of drug response. For the first time, we identify an isotopically unlabelled CYP3A4 substrate, tolterodine that leads to the formation of a non-toxic volatile metabolite, acetone, which could potentially be applied to monitor CYP3A4 activity in humans. In vitro biotransformation of tolterodine by HepG2 cells overexpressing CYP3A4, CYP2D6 or CYP2C9 was investigated by LC-MS analysis of cell culture supernatant for the non-volatile metabolite, N-dealkylated tolterodine, and PTR-ToF-MS analysis of the headspace for acetone. The highest level of the N-dealkylated metabolite was produced by HepG2-CYP3A4. Concentration dependent effects of tolterodine were analysed, resulting in TC50 values of 414 µM and 375 µM for HepG2-CYP3A4 and reference cells, respectively. Acetone and N-dealkylated tolterodine levels increased continuously over 24 h in HepG2-CYP3A4. Treatment with either a pan-CYP inhibitor, 1-aminobenzotriazole, or a CYP3A4 inhibitor, ketoconazole, considerably reduced the production of both metabolites in HepG2-CYP3A4 cells. These findings pave the way for the further development of non-invasive breath tests using unlabelled precursors to determine CYP enzyme activity in individuals.