Almost half of prescription medications are metabolized by cytochrome P450 3A4 and 3A5. CYP3A4 and 3A5 have significant substrate overlap and there is currently no way to selectively monitor the activity of these two enzymes individually, which has led to the erroneous habit of attributing the cumulative activity to CYP3A4. While CYP3A4 expression is ubiquitous, CYP3A5 expression is polymorphic, leading to individuals with large differences in CYP3A5 expression levels which have been shown to alter the pharmacokinetics of drugs in patients. We report the first highly selective CYP3A5 substrate, T-5, capable of determining CYP3A5 activity in biological samples containing both enzymes. Oxidation of T-5 by CYP3A5 yields an N-oxide metabolite that is over 100-fold selective over CYP3A4. Formation of T-5 N-oxide highly correlates with CYP3A5 genotype and CYP3A5 expression levels in human liver microsomes and human hepatocytes.