Abstract: Sample of MS culture medium had been mashed and mixed, and an aliquot (10 g) was taken and mixed with 10 mL of acetonitrile. The mixture was shaken for 20 min, and 1.14 g of sodium chloride and 2.24 g of anhydrous sodium sulfate were added. The mixture was shaken for 20 min, and centrifuged for 5 min. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was introduced into the high performance liquid chomatograph. Separation was carried out on the InertSustain C₁₈ column (150 mm×4.6 mm, 5 μm) by gradient elution with mixed solutions composed of acetonitrile and phosphate buffer solution (pH 3.5) at various volume ratios, and 6-benzylaminopurine, naphthalene acetic acid and indole butyric acid were detcted at detection wavelengths of 272, 221, 223 nm, respectively. It was shown that linearity relationships between values of mass concentration and peak area of the 3 plant growth regulators were kept in the range of 0.05-28.00 mg·L^-1, with detection limits (3S/N) in the range of 0.044 5-0.085 5 mg·kg^-1. Test for recovery was made by standard addition method, giving values of recovery in the range of 97.3%-103%, and RSDs (n=5) of the determined values ranged from 0.42% to 2.2%. After the culture medium was sterilized at 121 ℃ for 20 min, there was no significant change in the detection amounts of 6-BA and NAA compared with those before sterilization. The detection amount of IBA decreased obviously after sterilization, which reduced amounts of the detection amount after sterilization had linear positive correlation with detection amounts before sterilization.