A method was developed for the determination of 33 kinds of steroid hormones（including androgens,estrogens,progestogens,corticosteroids and steroids-like chemicals） in porcine plasma.The analytes in samples were extracted with methyl tert-butyl ether by vortex oscillation.The separation of analytes was achieved by gradient elution on a Xselect-C18 column（3.5 μm,2.1 mm×100 mm） under positive mode and an Extend-C18 column（3.5 μm,2.1 mm×100 mm） under negative mode.All steroids were detected by high performance liquid chromatography combined with tandem mass spectrometry（HPLC-MS/MS） and quantified by the external standard method.The calibration curves of 33 steroids were linear in the certain concentration range with correlation coefficients（r） more than 0.99.The average recoveries of steroids at three spiked levels ranged from 50.9% to 119.5% with relative standard deviations（RSDs） of 2.3%-9.8%.The limits of quantitation（LOQ,S/N≥10） were in the range of 0.05-40.00 ng/mL.The method was used for the determination of steroids in real samples collected from the large scaled livestock farms in Beijing and 10 kinds of steroids（including testosterone,progesterone,cortisone,androstenone,hydrocortisone,4-androstenedione,corticosterone,cortexolone,21-hydroxyprogesterone and 17α-hydroxyprogesteron） were detected.The results showed that the method was simple,sensitive and reproducible,and could be applied in the determination of steroids in porcine plasma.