Acorus tatarinowii Schott (ATS) is a well-known traditional Chinese medicine (TCM) for the treatment of epilepsy, amnesia and insomnia. In this study, a methodology utilizing high-performance liquid chromatography (HPLC) coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS/MS) was established for the separation and structural identification of the major chemical constituents in ATS for the first time. Overall, 46 major constituents including flavonoid glycosides, phenylpropane derivatives, amides and lignans were identified or tentatively characterized. Seven major constituents, including four phenylpropane derivatives and three lignans, were further quantified as marker substances, which showed good linearity within the test ranges. These results indicated that the developed quantitative method was linear, sensitive, and precise for quality control of ATS.