Detection, identification, and quantitation of human metabolites of new chemical entities represent an important component of the drug development process. Human metabolites that can raise a safety concern are those formed at >10% of total drug-related systemic exposure at steady state and that are not covered by the pre-clinical toxicology species. Performing human in vivo metabolite evaluation as early as possible during the drug development process can be very valuable. There are various challenges to obtaining this data, which include lack of metabolite reference standards and time and cost of a suitable 14C-labeled compound for and conducting an in vivo human absorption, metabolism and excretion (AME) study. Sparsentan is a dual antagonist of both the angiotensin 1 type 2 receptor and the endothelin A receptor and is currently in clinical development for its potential to treat patients with FSGS. To obtain an early measure of circulating metabolites of sparsentan, we compared the results of the metabolites identified in the plasma of patients with FSGS receiving sparsentan in the phase 2 DUET trial to 14C-sparsentan that was incubated with human liver microsomes or human hepatocytes to generate the 14C labelled metabolites of sparsentan. DUET, an 8-week, phase 2, double-blind, active-controlled study with an open-label extension, evaluated the efficacy and safety of sparsentan as a treatment for primary FSGS. Sparsentan and its 13 measurable metabolites generated in the in vitro systems were diluted into pre-dose plasma samples from patients and response factors for the individual metabolites were determined by comparing the radioactivity vs Tandem Mass Spectrometry signal intensity. These response factors were then applied to the metabolites identified in the FSGS patients who received sparsentan. The results indicated that unchanged sparsentan accounted for 85-87% of the circulating drug related material, while two mono-oxygenated metabolites composed 2-3% and 5-6% of the drug related material in plasma. These studies showed that there were no >10% disproportionate human metabolites following treatment with sparsentan.