Abstract: OBJECTIVE To establish a pre-column derivatization HPLC method which was used for determining the busulfan concentration in serum and pharmacokinetic characteristic of rabbits. METHODS 1,5-pentanediol dimethanesulfonate was used as internal standard, sodium diethyldithiocarbamate was used as derivatization reagent, and the derivatives of the busulfan and the internal standard were eluted with methanol-water (54:46) as mobile phase at a flow rate of 1.0 mL·min^-1 (0-20 min), 1.3 mL·min^-1 (20-27 min). The column temperature was 30℃, the detective wavelength was 280 nm and the injection volume was 25 μL. The rabbits were given busulfan by intragastric administration and intravenous injection respectively. The busulfan concentration in serum were measured by this method and the DAS 3.0 software was used to calculate pharmacokinetic parameters. RESULTS The blood concentration of busulfan was linear in the range of 0.1-3.4 mg·L^-1 (r=0.999 7). The intraday, interday precision and sample stability were in accordance with the 2015 edition of the Chinese Pharmacopoeia. The extraction recoveries of low, medium and high concentrations were 90.0%, 89.0% and 91.5%, respectively. The pharmacokinetic parameters of single dose orally were:t_1/2= (2.26±0.66)h, k= (0.33±0.12)·h^-1, k_a= (2.54±1.3)·h^-1, AUC_0-t= (1.95±0.18)h·mg·mL^-1; those of intravenous injection were:t_1/2= (1.53±0.09)h, k= (0.45±0.03)·h^-1, AUC_0-∞= (4.38±0.26)h·mg·mL^-1 and those of multi-dose oral administration were C_ss= (0.48±0.03)mg·mL^-1, AUC_0-τ= (3.87±0.26)h·mg·mL^-1. CONCLUSION The established method is suitable for determining the concentration of busulfan in serum and studying the pharmacokinetics. The pharmacokinetic parameters of the different routes of administration provide a practical basis for clinical pharmacokinetic studies.