Abstract: The medicinal nature of traditional Chinese medicine (TCM) mainly plays a role in regulating the energy and material metabolism of the body. The energy metabolism of the body is also regulated by the nervous and endocrine systems, and the hormones are an important component of the endocrine system to realize the coordinated action. Therefore, the medicinal nature of TCM is inextricably linked with the metabolism of hormones. Hyperthyroidism, qi-yin deficiency syndrome is regarded as the classic heat syndrome category in Chinese medicine, which can be used as model to reflect the mechanism and difference of qi tonifying of ginseng herbs with different medicinal natures. Steroid hormones are synthesized in the adrenal cortex, mitochondria of placental cells, smooth endoplasmic reticulum, and gonads of the human body by the participation of various metabolic enzymes. Meanwhile, most of the patients with hyperthyroidism have adrenocortical dysfunction in the clinic, and the corresponding clinical symptoms are closely related to the hypothalamic-pituitary-thyroid axis (HPT), the hypothalamic-pituitary-adrenal axis (HPA), and the hypothalamic-pituitary-gonadal axis (HPG). When hyperthyroidism occurs, the production and release of steroid hormones will be affected to a certain extent. To explore the correlation between the level of steroid hormones and hyperthyroidism, and the ameliorative effects of red ginseng and ginseng leaves of different types of the genus Ginseng on hypermetabolism of hyperthyroidism and the changes of related indexes, a method of ultra-high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) was established for rapid identification and quantification of chemical components, including adrenaline, androstenedione, testosterone, dehydroepiandrosterone, estriol, progesterone, 17α-hydroxypregnenolone, corticosterone, and cortisol in rat serum. Then, multivariate analysis method, principal component analysis (PCA), and Spearman test were used to analyze the correlation among adrenaline, steroid hormones, and thyroid function. The results showed that the contents of steroid hormone in the serum of the model group of rats decrease to different degrees, compared with the control group, and increase after the administration of different types of ginsengs. Meanwhile, it was proved that the established method has high sensitivity, good reproducibility, high accuracy, and fast analysis, which can screen a large number of steroid hormones in complex samples in a short time, for accurately investigation of the effects of ginsengs on the content of 9 types of steroid hormones in hyperthyroidism rats. The abnormal levels of steroid hormones are related to thyroid hormones. Corticosterone and adrenaline can serve as key indicators for regulating steroid hormone metabolism of TCMs like ginsengs. Testosterone can be used as the biomarker for regulating steroid hormones of the cold nature herbs. Adrenaline and androstenedione can be used as biomarkers for regulating steroid hormones of the warm nature herbs. This method can conveniently, quickly, accurately, and efficiently complete the analysis of target components in samples. The average recoveries of steroid hormones in rat serum range from 88.75% to 106.94%, and the relative standard deviation is less than 1.99%. This can be used for the determination of steroid hormones in rat serum systematically, rapidly, accurately and efficiently, and provides a methodological reference for the analysis and determination of steroid hormones.