Quality marker (Q-Marker) is an innovative concept and model for quality control of Traditional Chinese medicines (TCMs), which will navigate the new direction of quality development of TCMs. Yet, how to characterize the overall quality attributes of TCMs and their biological effects is still debating. In view of this key scientific issue, this paper proposes a research method based on mass spectrometry imaging (MSI) technology for the discovery and confirmation of TCMs Q-Marker. MSI is powerful in investigating the spatial distribution of molecules in a variety of samples, and visualizing the information obtained from MS. On this basis, combine with the five principles of TCMs Q-Marker validation, i.e., specificity, transmission and traceability, testability, prescription compatibility, and validity, were applied to confirm the finalized Q-Marker. It will lead the new direction of quality development of TCMs.