OBJECTIVE： To establish a method for the concentration determination of indigo in rats’ plasma， and study the pharmacokinetic characteristics in rats in vivo. METHODS： 18 rats were randomly divided into low-dose， medium-dose， high-dose groups， 6 in each group， which were intragastrically administrated 10， 20， 40 mg/kg of indigo solution. The sample blood 0.3 mL was taken from eye socket before administration and 0.083， 0.25， 0.5， 0.75， 1， 2， 4， 6， 8， 10， 12， 16， 24， 48， 72 h after administration， separating the plasma， then LC-MS/MS was used to determine the plasma concentration of indigo after methanol precipitation. The column was Agilent Poroshell EC-C18 with mobile phase consisting of methanol-5 mmol/L ammonium acetate solution （95 ∶ 5，V/V） at a flow rate of 0.4 mL/min； multiple reaction monitoring was conducted for the quantitative analysis， with ion pair of 263.1-218.8 （indigo） and 237.2-194.1 （carbamazepine， internal standard）. Pharmacokinetic parameters were calculated by DAS 3.0 software. RESULTS： The linear range of indigo was 0.5-100 ng/mL （r＝0.999 9）， intra-day and inter-day RSDs were lower than 9％ （n＝5）； matrix effects of low， medium and high does quality control samples were （98.25±3.71）％， （102.23±2.64）％， （102.29±3.79）％ （n＝5）. The pharmacokinetic parameters of indigo in low-dose， medium-dose， high-dose groups were tmax of （8.6±1.1）， （9.2±0.8） and （9.5±0.8） h； cmax of （30.9±8.6）， （44.9±10.1）， （96.1±17.4） ng/mL； t1/2 of （14.9±2.1）， （16.3±2.9）， （15.3±3.7） h； AUC0-72 h of （366.6±83.4）， （694.9±105.8）， （1 223.42±108.7） ng·h/mL， respectively. CONCLUSIONS： The method shows high sensitivity， good specificity， and can be used for the content determination of indigo in plasma samples of rats. The pharmacokinetics of indigo in rats in vivo fits non-compartment model.