Abstract: The present study was designed to identify and characterize the major constituents in Juglans mandshurica Maxim. A simple, efficient and sensitive ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-ESI-Q-TOF/MS) method was established and validated under positive and negative ion modes. The separation was performed on a Waters ACQUITY UPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) by gradient elution with a mobile phase (Phase A:0.1% aqueous formic acid solution, Phase B:0.1% formic acid acetonitrile solution). A total of 165 compounds were rapidly selected by Targeted and Non-Targeted Peak Finding approaches, and then tentatively identified by comparing with reference substances or inferred through mass spectrometry fragment ion analysis and literature data. These compounds included 68 naphthalenequinones, 20 diarylheptanoids, 29 flavonoids, 20 triterpenes, and 28 phenolic acids. In conclusion, the present study provided an effective approach to identifying components in complex matrices of herbal medicines such as Juglans mandshurica Maxim.