Background: Danmu injection, a traditional Chinese medicine (TCM) preparation made from Nauclea officinalis, has been commonly used for the treatment of cold, fever, swelling of throat in China. However, the chemical constituents in Danmu injection have not been clarified yet., Objective: a HPLC/DAD/ESI-MS(n) method was developed for qualitative and quantitative analysis of the components in Danmu injection., Materials and Methods: The chromatographic separation was performed on a Welch Material XB-C18 (4.6mm × 250mm, 5μm) using gradient elution with acetonitrile (A) and water containing 0.1% formic acid (B) as mobile phase at a flow rate of 1.0 ml/min., Results: Twenty-five compounds, including phenolic acid and phenol glycoside, iridous glycoside and glycoalkaloid were identified or tentatively deduced on the base of their retention behaviors, UV absorption, MS and MS(n) data with those elucidated references or literature. In addition, eleven compounds were simultaneously determined by HPLC-DAD, which was validated and successfully applied for determination of major components in Danmu injection., Conclusion: The results suggested that the established qualitative and quantitative method would be a powerful and reliable analytical tool for the characterization of multi-constituent in complex chemical system and quality control of Danmu injection.