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 I3 C, DIM, and other I3 C metabolites in plasma. Samples containing I3 C 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 I3 C 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 m L/min and the run time was 9 min. The limits of detection and quantification for I3 C and DIM were 15 ng/m L and 25 ng/m L, respectively. Calibration curves for I3 C and DIM were linear(r2>0.99) over a concentration range of 0.025–20 μg/m L. Precision, accuracy, and stability analysis fulfilled the CDER guidelines criteria. The method was successfully applied to the determination of the pharmacokinetic parameters of I3 C or DIM after oral, intravenous, or intraperitoneal administration to Sprague Dawley rats. The method described here is superior over existing analytical methods for I3 C and its metabolites in terms of sensitivity, speed, and separation. 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. 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 I3 C, DIM, and other I3 C metabolites in plasma. Samples containing I3 C or DIM and the internal standard 4-methoxy The mean recovery was 96.21% for I3 C 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 m L / min and the run time was 9 min. The limits of detection and quantification for I3 C and DIM were 15 ng / m L and 25 ng / m L, respectively. Calibration curves for I3 C and DIM were linear (r2> 0.99) over a concentration range of 0.025-20 μg / m L. Precision, accuracy, and stability analysis fulfilled the CDER guidelines criteria. The method was successfully applied to the determination of the pharmacokinetic parameters of I3 C or DIM after oral, intravenous, or intraperitoneal administration to Sprague Dawley rats. The method described here is superior over existing analytical methods for I3 C and its metabolites in terms of sensitivity, speed, and separation.