An efficient enantioselective method for the determination of etoxazole in orange pulp, peel, and whole orange was developed using liquid chromatography with tandem mass spectrometry. The enantioseparation was performed on a Chiralpak AD-3R column at 30ºC using acetonitrile with 0.1% formic acid solution (80:20, v/v) as the mobile phase in less than 5 min. Quantification was achieved using matrix-matched standard calibration curves. The overall mean recoveries for two enantiomers from orange pulp and whole orange were 91.0-99.6% and the orange peel was 92.6-103.1%, with relative standard deviations of 0.8-5.4% intraday and 2.0-4.8% interday at 1, 10, and 100 μg/kg levels, and 1.3-5.2% intraday and 3.5-4.3% interday at 5, 50, and 500 μg/kg levels, respectively. The limits of quantification for all enantiomers in three matrices did not exceed 5 μg/kg. Moreover, the absolute configuration of etoxazole enantiomers had been determined by the combination of experimental and predicted electronic circular dichroism spectra, and the first eluted enantiomer was confirmed as (S)-etoxazole on a Chiralpak AD-3R column while (R)-etoxazole was first on three cellulose chiral columns. The application of the proposed method to real sample analysis suggests its potential use in enantioselective determination of etoxazole enantiomers in citrus.