Abstract: Due to the composition complexity of traditional Chinese medicines (TCMs), the identification and clarification of the chemical components of TCMs plays a crucial role in the evaluation of safety and efficacy of their clinical use. In this work, an ultrahigh performance liquid chromatography-quadrupole-electrostatic field Orbitrap mass spectrometry (UHPLC-Q-Orbitrap/MS) was used to analyze the chemical composition of the substance benchmark of ZhuYe ShiGao decoction (ZSD), a widely used TCM formula. The UHPLC separation was performed on a SUPELCO C18 column (100 mm×4.6 mm×2.7 μm) with water (A) and acetonitrile (B) both containing 0.1% formic acid as mobile phases. The column temperature was 30 ℃, and the injection volume was 5 μL. The gradient started with 5%B, and increased to 20%B at 5 min, to 40%B at 15 min, to 95%B at 25 min, dropped back down to 5%B at 27 min, and remained 5%B to 35 min with a flow rate of 0.5 mL/min. The molecular ions and fragment ions data obtained by HRMS in positive and negative ion modes were used to identify the components in the substance benchmark of ZSD by referring to the self-constructed database from the related literatures and the MS data collected from control products, and to study the MS fragmentation mechanism. A total of 121 compounds were identified, including 47 triterpenes, 34 flavonoids, 10 steroidal saponins, and 30 other compounds. Among them, the flavonoids are mainly from Lophatheri, Glycyrrhizae and Ophiopogonis, the saponins are mainly from Ginseng and Glycyrrhizae, and the alkaloids are mainly from Pinelliae. ZSD formula consists of 7 herbs, of which the components may be loss during the preparation of the substance benchmark. For example, the amino acids in Pinelliae was significantly reduced in the substance benchmark, and the widespread presence of steroidal saponins in Ophiopogonis was also converted during the preparation. In addition to the loss of components, components in different herbs may react with each another to produce new components during the preparation of the substance benchmark. These not only led to absence of identification of some well-known components in the herbs of ZSD, but also made it difficult to identify the newly produced compounds with high intensity of MS signals, requiring further study. Even so, however, the present work identified the chemical composition of the ZSD substance benchmark and explored the fragmentation pathways of the identified components by using UHPLC-Q-Otbitrap/MS, providing a good reference basis for quality control and the pharmacodynamics research of ZSD.