Chinese materia medica processing is a distinguished and unique pharmaceutical technique in traditional Chinese medicine (TCM), which has played an important role in reducing side effects, increasing medical potencies, altering the properties and even changing the curative effects of raw herbs. The efficacy improvement in medicinal plants is mainly caused by changes in the key substances through an optimized processing procedure. Thus, the use of a rapid method for determining suitable chemical markers between raw and processed TCM is critical in order to elucidate how the bioactive compounds influence the clinical effects. In this study, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry combined with MS/MS-based molecular networking (MN) and a multivariate statistical analysis method is proposed for the first time. This combination was used to identify the complex chemical composition and clarify the changed constituents between raw and processed Cistanche tubulosa (C. tubulosa). The chemical analysis results demonstrated that a total of 85 compounds were identified in the crude and processed C. tubulosa. Moreover, 34 compounds were detected as chemical markers. This systematic research into chemical constituents and chemical markers of crude and processed C. tubulosa lays a solid foundation for further study of the quality control of C. tubulosa. Moreover, the study provides a new and valuable technical strategy for analyzing chemical components and identifying potential chemical markers for the processing of herbal medicines.Graphical abstract.