A liquid chromatography-tandem mass spectrometric（LC-MS/MS） method for the determination of corticosterone and hydrocortisone in serum was developed.The fat in serum was removed with hexane and extracted with methyl ether.Corticosterone and hydrocortisone were separated on an Agilent Eclipse Plus-C18 column（150 mm×2.1 mm，3.5 μm） and detected by chromatography coupled with tandem mass spectrometry in positive ion mode with mobile phase of acetonitrile-0.1% formic acid（containing 0.01 mol/L ammonium formate）.The flow rate of mobile phase was set at 0.3 mL/min.The calibration curves of two target compounds were in the range of 0.5-200 ng/mL，with correlation coefficients（r2） of 0.999 7 and 0.999 4，respectively.The recoveries for the spiked sample at the levels of 5.0，25.0 and 75.0 ng/mL for corticosterone and 0.5，2.5,10.0 ng/mL for hydrocortisone，ranged from 86.6 % to 102.7% with relative standard deviations not more than 7.1%.The limits of detection（S/N=3） for corticosterone and hydrocortisone were 0.1 ng/mL and 0.05 ng/mL，respectively.This method was simple，sensitive and accurate，and was suitable for the determination of corticosterone and hydrocortisone in serum sample.