The purpose of this research was to explore the differences of the components of Radix Polygalae in herbal pair of Radix Polygalae and Rhizoma Acori Tatarinowii. An HPLC-Q-TOF-MS/MS method and an HPLC-UV method were established for the identification and determination of the components of Radix Polygalae, respectively. HPLC separation was carried out on a C18 column(250 mm×4. 6 mm, 5 μm)with linear gradient elution using a mobile phase consisting of acetonitrile and 0. 1% formic acid aqueous solution. Mass spectrometry with ESI source was performed in the positive ion mode to scan MS data including total ion chromatograms and ion peaks of Radix Polygalae. Eight components including 3, 4, 5-trimethoxycinnamic acid, p-methoxycinnamic acid, tenuifolin, sibiricose A5, polygalaxanthone III, tenuifoliside B, 3, 6′-disinapoly sucrose, and tenuifoliside A were identified according to the reference substance retention time, MS data and literatures. There was no significant variation found in the contents of eight chemical constituents of Radix Polygalae. The qualification and quantitation study of the components in herbal pair of Radix Polygalae and Rhizoma Acori Tatarinowii provide the methodological basis for compatibility mechanism exploration in vivo.