Abstract: A method of ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) was developed to determine the contents of deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, cholic acid, glycinecholic acid, taurocholic acid, tauroporic deoxycholic acid, taurocholic acid in the serum of rats with hyperthyroidism and each administration group. The separation of compounds was carried out on an Agilent zorbax SB-C18 column (100 mm×2.1 mm×1.8 μm). The mobile phase was 0.1% formic acid in acetonitrile and 0.1% formic acid in the water, the flow rate was 0.4 mL/min, the injection volume was 5 μL. The mass spectrometry was determined by electrospray ionization in negative ion mode, and was scanned by multiple response monitoring mode. The ion scanning range was m/z 50-1 000, with a capillary voltage of 3.5 kV, ion source temperature of 150 ℃, atomization gas flow rate of 50 L/h, desolvation gas flow rate of 800 L/h, desolvation temperature of 250 ℃. Multivariate analysis method of principal component analysis (PCA) was used to analyze the content of bile acids in each group, which identified the nature of ginseng and biomarkers of bile acids that regulated the cold and hot nature of ginseng. Spearman was used to analyze the correlation between bile acid level and thyroid function. The results showed that the calibration curves of 8 bile acids were linear in the quantitative range. The average recoveries of bile acid isotopes ranged from 82.71% to 104.05% and the coefficients of variation were all less than 15%. Compared with the blank control group, the contents of deoxycholic acid, goose deoxycholic acid, porcine deoxycholic acid, and cholic acid in the serum of hyperthyroidism rats decreased, and the content of primary bile acid-cholic acid increased after administration of Phellodendrina chinense and ginseng leaves. The cholic acid, deoxycholic acid, and goose deoxycholic acid can be used as the biomarkers for regulating bile acids in cold and cool nature herbs, and glycine cholic acid, deoxycholic acid, and taurocholic acid can be used as the biomarkers for regulating bile acids in warm nature herbs. The level of thyroxine (T4) was negatively correlated with deoxycholic acid. The level of tri-iodothyronine (T3) was negatively correlated with porcine deoxycholic acid, glycine cholic acid, taurocholic acid, taurocholic acid, and taurocholic acid. The UPLC-QqQ-MS/MS method has the advantages of simple operation, high sensitivity, and strong multiplexing detection capability, which can be used to determine the content of serum bile acid. The abnormal level of bile acid is associated with thyroid hormone to a certain extent.