Abstract: A rapid identification of the constituents in Gualoupi injection was developed by HILIC/Orbitrap Fusion Lumos HRMS. ACQUITY XBridge Amide column was used to isolate the constituents with large polarity. ESI with positive ion mode was employed and "Top Speed" DDA was applied in the MS² scan. Compound identification was carried out by using Compound Discoverer software through comparing the precursor and product ions information with those in ChemSpider and mzCloud database. As a result, 48 compounds was identified, including alkaloids, amino acid, nucleosides, nucleobases, etc., among which 25 was unambiguously identified by comparing the retention time and mass spectra information with those of reference standards. In general, the main constituents from Gualoupi injection were explained in this study. Additionally, full-scan mass spectra of 21 batches of the injection were collected by the established method. Subsequently, principal component analysis (PCA) with the peak area as the observation ID was employed to analyze the discrimination of different bathes of samples. The results indicated that the batch-to-batch difference was generally from the crude drug, and the preparation process of this injection was relatively stable. In conclusion, a rapid and effective method for the identification of compounds in Gualoupi injection was established by using UHPLC-Orbitrap Fusion Lumos HRMS and the inter-bath stability was also investigated, which may make a great contribution to the study of the bioactive ingredients in Gualoupi injection and also provide vital evidence for the standard promotion.