AbstractA sensitive and simple high‐performance liquid chromatographic method with UV detection was developed and validated for the determination of andrographolide in rat whole blood. Carbamazepine was employed as internal standard and the blood sample was extracted with chloroform. Chromatographic separations were achieved on a Chromasil ODS column (250 × 4.6 mm, 5 µm). The mobile phase was consisted of methanol–water (52:48, v/v) and delivered at 0.8 mL/min. The detection wavelength was set at 225 nm. The calibration curve had a good linearity in the range 0.053–530 µg/mL in rat whole blood with its correlation coefficient being 0.996. The extraction recovery of andrographolide was ranged from 65.7 to 72.6%. The intra‐day and inter‐days repeatabilities were below 4.2% in terms of the percentage of relative standard deviation (RSD). The method was used to provide data on the pharmacokinetics of the drug in rats. The data obtained was processed using the 3P87 pharmacokinetic program. The results showed that the disposition of andrographolide after intravenous administration of liposomal andrographolide conformed to a two‐compartment open model with α = 4.75 × 10−2 ± 2.41 × 10−3 min−1, β = 3.16 × 10−3 ± 1.58 × 10−4 min−1, Vc = 174.67 ± 13.97 mL, k21 = 1.60 × 10−2 ± 8.12 × 10−4 min−1, k10 = 9.38 × 10−3 ± 5.62 × 10−4 min−1, k12 = 2.53 × 10−2 ± 1.27 × 10−3 min−1 and AUC0‐∞ = 1525.47 ± 92.35 µg min/mL. For the intragastric administration of andrographolide tablets, the disposition of andrographolide followed a one‐compartment open model with ke = 6.78 × 10−3 ± 3.53 × 10−4 min−1, ka = 3.69 × 10−2 ± 4.68 × 10−3 min−1, Tmax = 59.69 ± 3.61 min, Cmax = 1.62 ± 0.11 µg/mL, Vc = 1056.90 ± 83.42 mL, AUC0‐∞ = 348.75 ± 24.41 µg min/mL. Copyright © 2007 John Wiley & Sons, Ltd.