Evaluating drug metabolizing activity using probe drugs can be challenging and costly. Barriers to using probe drugs for phenotyping include: concerns about the side effects of the drug, invasiveness of sampling (e.g., repeated blood collection), duration of a clinical study day, and inability to easily conduct the study in vulnerable populations such as children and pregnant women. Using endogenous biomarkers might provide a simple, relatively non-invasive, safe, and time/cost-effective means of determining an individual’s phenotype. In recent years, research has focused on identifying endogenous biomarkers of CYP3A activity (e.g., 6β-hydroxycortisol, 4β-hydroxycholesterol, and other newly identified compounds). However, many gaps still exist. A thorough understanding of the “pharmacokinetics” of these endogenous compounds will permit more accurate interpretation of changes due to drug interactions. As discussed in this talk, there are several unique challenges with using endogenous biomarkers that need to be addressed before these compounds can replace traditional probe substrates.