Abstract: In the past decades, systematic isolation and structural identification of chemical components from plant material, especially traditional Chinese medicine, have provided a large number of chemical drugs and lead structures for clinic. However, there are still a large number of trace components in traditional Chinese medicine with novel chemical structures and specific pharmacological activities, which need to be further analyzed. With the development of modern analytical technology, the network technology based on tandem mass spectrometry and cluster analysis shows many advantages in trace analysis of complex components. In this paper, a method of high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS) was developed for the analysis of the main chemical constituents in the root bark of Lycium barbarum L. The aqueous extract sample was separated by methanol aqueous through C18-solid phase extraction, and then the 50% methanol aqueous elution was analyzed using HPLC-Q-TOF MS with C18 column. The ions were monitored in both positive mode and negative mode of ESI with the scanning ranges of m/z 100-300 and 300-2 000. The trace chemical components were analyzed by molecular network technology. The MS data files were converted to the mzXML format and the molecular network was generated on GNPS, then visualized with Cytoscape, and the structures of compounds were predicted based on literature data. Twenty one known compounds in Cortex Lycii were verified by manual analysis technique and eleven new components were firstly identified by molecular networking. It is worth reporting that compound 24 and 25 are different from cyclooctapeptides previously found in the root bark of Lycium chinense (another source of Cortex Lycii), indicating that they can be used as markers for the identification of two species of Cortex Lycii. This work lays a research foundation for promoting the effective application of Lycium barbarum L. root bark.