Olaquindox (OLA), N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide, is an antimicrobial and growth-promoting agent for animals, which has been banned or allowed only limited use for its potential toxicity. To thoroughly understand the metabolic pathways, metabolism of OLA in rat was studied using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with MSE and mass defect filtering techniques. Twenty metabolites (M1–M20) were detected in rat feces and urine, of which nine phase I metabolites (M6, M7, M11–M16) and four phase II metabolites (M17–M20) were found in vivo for the first time. The structures of metabolites were reliably characterized on the basis of accurate mass and fragment ions in MSE spectra. The major metabolic pathways reported previously in pigs, including reduction of N[RIGHTWARDS ARROW]O groups, oxidation of the alcohol and hydrolysis, were also confirmed in this study. In addition, hydroxylation of the methyl group, N-dehydroxyethylation and glucuronidation were also proved to be the important metabolic pathways, which contribute to improving our knowledge about in vivo metabolism of OLA. Copyright © 2011 John Wiley & Sons, Ltd.