Indole-3-carbinol (I3C) and diindolylmethane (DIM) are naturally derived dietary phytochemicals with promising anti-cancer properties that have been demonstrated both in vitro and in vivo. Using reversed-phase ultra-performance liquid chromatography(UPLC) coupled with mass spectrometry (MS), a rapid, specific, and high throughput method was developed and validated for the quantification and identification of I3C, DIM, and other I3C metabolites in plasma. Samples containing I3C or DIM and the internal standard 4-methoxy indole (IS) were extracted using a liquid-liquid extraction technique. The mean recovery was 96.21% for I3C and 108.5% for DIM. Separation was achieved using a Waters Acquity UPLC HSS T3, 1.8 µm, 2.1 mm×150 mm column and acetonitrile–water gradient elution. The flow rate was 0.3 mL/min and the run time was 9 min. The limits of detection and quantification for I3C and DIM were 15 ng/mL and 25 ng/mL, respectively. Calibration curves for I3C and DIM were linear (r2>0.99) over a concentration range of 0.025–20 µg/mL. Precision, accuracy, and stability analysis fulfilled the CDER guidelines criteria. The method was successfully applied to the determination of the pharmacokinetic parameters of I3C or DIM after oral, intravenous, or intraperitoneal administration to Sprague Dawley rats. The method described here is superior over existing analytical methods for I3C and its metabolites in terms of sensitivity, speed, and separation.