Abstract: Sterones are one of the active components of Achyranthes bidentata, and the difficulty in distinguishing sterone isomers poses a great challenge to the accurate identification of the chemical components of Achyranthes bidentata, while online energy-resolved mass spectrometry (Online ER-MS) has been proved to be an effective method for distinguishing the isomers due to its ability to provide rich MS/MS fragment information. In this study, mass spectra of sterones were collected by ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS), and then the structures of the compounds were matched with MS/MS fragment ions to initially identify the sterone compounds in Achyranthes bidentata. In order to further distinguish the sterones isomers, the fragment ions with higher abundance detected in UHPLC-Q-TOF-MS/MS were selected to construct pseudo-ion transitions (PITs), and the collision energy (CE) and relative ion intensity of PITs were collected by using the multi-reaction monitoring mode of ultra-high performance liquid chromatography tandem quadrupole linear ion trap mass/mass spectrometry (UHPLC-Q-trap-MS/MS). Further, the relative ion intensity of each PIT under different CE was recorded, and the dataset was imported into GraphPad Prism software to draw the fragmentation curve for each ion transition via Gaussian fitting. The vertex of the fragmentation curve was defined as the optimal collision energy (OCE), and the OCE of different ion transitions of each compound was calculated respectively. At the same time, combined with quantum structural calculation, the thermal enthalpies of precursor ions and product ions in each candidate structure were calculated using Gaussian program based on density functional theory, and the bond energy required to break the corresponding chemical bond was calculated according to the difference between the precursor ion and the two product ions. The OCE and bond energies of each group of isomers were analyzed to establish the relationship between the chemical structure and OCE. The results showed that the energy required to break the same chemical bond was not the same due to the different substituent positions of isomers, which made the OCE of different ion transitions of isomers also differ. Finally, 12 sterones including three groups of the isomers were identified from Achyranthes bidentata, and the feasibility of the method was verified by comparing with the standards. Therefore, the combination of online energy-resolved mass spectrometry and quantum structural calculation can achieve efficient differentiation of isomers, providing a new solution for accurate identification of isomers in traditional Chinese medicines.