Rilpivirine is a nonnucleoside reverse transcriptase inhibitor used to treat HIV-1. In the present study, the pathways responsible for the biotransformation of rilpivirine were defined. Using human liver microsomes, the formation of two mono- and two dioxygenated metabolites were detected via ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Mass spectral analysis of the products suggested that these metabolites resulted from oxygenation of the 2,6-dimethylphenyl ring and methyl groups of rilpivirine. Chemical inhibition studies and cDNA-expressed cytochrome P450 (CYP) assays indicated that oxygenations were catalyzed primarily by CYP3A4 and CYP3A5. Glucuronide conjugates of rilpivirine and a monomethylhydroxylated metabolite of rilpivirine were also detected and were found to be formed by UDP-glucuronosyltransferases (UGTs) UGT1A4 and UGT1A1, respectively. All metabolites that were identified in vitro were detectable in vivo. Further, targeted UHPLC-MS/MS-based in vivo metabolomics screening revealed that rilpivirine treatment versus efavirenz treatment may result in differential levels of endogenous metabolites, including tyrosine, homocysteine, and adenosine. Rilpivirine biotransformation was also assessed across species using liver microsomes isolated from a range of mammals, and the metabolite profile identified using human liver microsomes was largely conserved for both oxidative and glucuronide metabolite formation. These studies provide novel insight into the metabolism of rilpivirine and the potential differential effects of rilpivirine- and efavirenz-containing antiretroviral regimens on the endogenous metabolome.