A high performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry method was developed for the identification of phenolic compounds in Vitex negundo L. var. cannabifolia (Siebold et Zucc.) Hand.-Mazz. A total of 31 compounds (10 phenolic acids, 19 flavonoids, and 2 iridoids) were fully or partially identified. Caffeic acid, neochlorogenic acid, cryptochlorogenin acid, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, schaftoside, isoschaftoside, flavosativaside, vitexin 2″-rhamnoside, and kaempferol 3-(6″-malonylglucoside) were detected for the first time in this plant. In subsequent quantitative analysis, an ultra-performance liquid chromatography combined with triple quadrupole mass spectrometry method was developed for the quantitative analysis of 17 phenolic compounds. All the analytes were detected within 8. min. The limits of detection and quantification were less than 7.251 and 26.454. ng/mL, respectively. The relative standard deviations of intra-day precision, inter-day precision, repeatability, stability, and recovery were less than 2.87%, 3.87%, 4.86%, 4.70%, and 3.61%, respectively. The validated method was applied to assess the quality of different medicinal parts (leaves, seeds, and roots) of V. negundo. The results indicated that chlorogenic acid, agnuside, isochlorogenic acid A, and isochlorogenic acid C might be selected as quantitative markers for the quality control of V. negundo. The contents of the 17 investigated compounds in leaves differed from those in the other parts. Hierarchical cluster analysis suggested that chlorogenic acid, agnuside, vitexin, and schaftoside could be potential chemical markers for the discrimination of different medicinal parts of V. negundo. This qualitative and quantitative analysis of phenolic compounds of V. negundo could provide a new tool for the quality control of this plant or its related remedies. © 2015 Elsevier B.V.