Abstract: AimTo develop an HPLC method for the determination of Aconitum alkaloids extracted from Radix aconiti preparata in rats. MethodsWaters 2690@996 PAD system was used. The analytical column was a Halsil 100 C₁₈ column (250 mm×4.6 mm ID, 5 μm). The mobile phase was water, methanol and diethyl amine at the ratio of 75∶25∶0.1. The flow rate was 0.9 mL·min^-1. The wavelength of the detector was 240 nm. ResultsThe linear ranges of aconitine in the heart, spleen, lung and kidney were 0.4-100 μg·mL^-1, the correlation coefficients were 0.997 2, 0.998 6, 0.999 3 and 0.999 4, respectively. The linear range of aconitine in liver was 2-200 μg·mL^-1 and the correlation coefficient was 0999 0. The linear ranges of hypaconitine in heart, liver, spleen, lung, kidney, brain and spinal cord were 5-100 μg·mL^-1, the correlation coefficients were 0.999 4, 0.999 7, 0.999 8, 0.998 4, 0.999 8, 0.999 8 and 0.999 7, respectively. Detection limits (S/N=3) of aconitine and hypaconitine were 0.4 μg·mL^-1. The recoveries of aconitine and hypaconitine ranged from 88.7% to 102.2% and 86.5% to 101.3%, respectively, and the RSD of precision of aconitine and hypaconitine was 10%. ConclusionIt appears to be an accurate and effective method that can offer reference basis for in toxication of Radix aconiti preparata clinically.