AIM: To analyze the major constituents in Radix Scrophulariae(Scrophularia ningpoensis). METHOD: Radix Scrophulariae was analyzed by HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry(ESI-Q-TOF MS/MS). Compounds were separated by HPLC using a C18 column and gradient elution of acetonitrile and 0.1 %(V/V) acetic acid-water. Negative ion mode was employed. RESULTS: A total of thirty-six compounds, including fourteen iridoid glycosides, nineteen phenylpropanoid glycosides, and three organic acids, were identified from Radix Scrophulariae based on the accurate mass measurement of precursor and product ions. Twenty-one of the constituents were identified by comparing their retention times(tR) and ESI-MS/MS data with those of reference standards and/or previous publications, while another fifteen compounds were tentatively identified or deduced according to their Q-TOF MS/MS data which afforded sufficient structural information. CONCLUSION: It is believed that this study is useful for the identification of constituents in Radix Scrophulariae, as well as related plants and complex prescriptions.