Abstract: A method for the determination of 20-hydroxyecdysone in hemolymph of Bombyx mori was developed by high performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）. 22(S), 23(S) homobrassinolide was used as an internal standard, and acetonitrile was used to precipitate the proteins in the hemolymph of Bombyx mori to eliminate the interference. The gradient elution program was performed on a C18 column using a mobile phase consisting of acetonitrile and 0.5% acetic acid solution. Under the optimum working conditions of the instrument，the analytical results show that the recoveries of 20-hydroxyecdysone in hemolymph of Bombyx mori are in the range of 83.56%-97.11%， and the relative standard deviation is below 5.4%. Linearity of the method in the range 3.13-50 μg/L is good with the correlation coefficients(R²) of 0.9963. The detection limit of the method is 0.2 μg/L. The method can meet the need for the determination of 20-hydroxyecdysone in the hemolymph of Bombyx mori, which may also be applied to determine 20-hydroxyecdysone in the hemolymph of other insects.