Abstract: Mulberry has been widely researched for its unique health care effect. Its 85% ethanol extract is included in the Chinese Pharmacopoeia. A method for analyzing the flavonoids and polyphenols of mulberry extracts was developed by high-performance liquid chromatography quadrupole-Orbitrap mass spectrometry. Most studies of mulberry focused on the content determination, extraction and separation, pharmacological activities, while the analysis of active ingredients in mulberry flavonoids was seldom investigated. Studies have shown that the total content of flavonoids in the mulberry is 1.508 mg/g. The research of flavonoids and polyphenols is of great significance to control the quality of medicinal and edible mulberry as well as to develop the valuable source reasonably. Recently, the HPLC/MS has been proved as an effective method for component analysis in complex extracts system, through which the compounds in mixtures and yield information based on their molecular weights as well as the structures can be rapidly determined. In this study, high-performance liquid chromatography quadrupole-Orbitrap mass spectrometry was adopted for its high speed, high resolution and high sensitivity. It is also effective for compound structure identification online. Analysis of flavonids and polyphenols was performed using H₂O-CH₃CN containing 0.1% formic acid with 0.2 mL/min flow rate by a Syncronis C18 column (100 mm×2.1 mm, 1.7 μm). Five flavonoids of rutin, isoquercerin, kaempferol-7-glucoside, taxifolin, quercetin and three polyphenols of 3,4-dihydroxyphenylacetic acid, chlorogenic acid, caffeic acid are identified by retention time, accurate molecular weight, tandem mass spectrometry fragmentation information. The retention times of these eight identified compounds are 7.08, 8.32, 9.05, 9.50，9.88, 10.33 and 13.46 min, respectively. In full MS scan, these eight compounds give M-H^- ion at m/z 153, 353, 179, 609, 463, 447, 303 and 301 in negative ion mode. For the further analysis to identify the structures of these compounds, they were subjected for the tandem mass spectrometry. MS fragmentation patterns of compounds were discussed and it provided basis for their structure identification. Loss of glucose residue (162 u), CO (28 u), CO₂ (44 u) and H₂O (18 u) are characteristic losses for the identification of flavonoids in mulberry which offer the important structural information. This method is rapid, simple, accurate, which can provide chemical foundations for exploitation of mulberry in drugs and foods reasonably.