Microctis Folium (MF), the leaves of Microcos paniculata L., which is widely used as a traditional Chinese medicine and a healthy tea, show various biological activities including antioxidant, cardiovascular risk, and so on. However, few studies on the quality control for this herbal medicine have been reported. In this paper, a reliable and sensitive online high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem ion trap mass spectrometry (HPLC-DAD-ESI-MS/MS) method for qualitative and quantitative evaluation of the characteristic chemical constituents of MF was developed. 32 compounds including 10 flavans, 16 flavonoid glycosides, 5 lignan glycosides and 1 megastigmadien glycoside were identified from MF, among which lignan glycosides and megastigmadien glycoside were first reported from the genus Microcos. Besides, based on HPLC-DAD-ESI-MS/MS data, a standardized HPLC fingerprint and a quantitative method were established by analyzing 13 batches of commercial herbal samples by HPLC with ultraviolet-visible (UV) detection method. Meanwhile, three major bioactive constituents including kaempferol-3-O-β-D-glucoside, narcissi and isorhamnetin-3-O-β-D-glucoside in MF were simultaneously determined with the same method, which could differentiate good quality batches from counterfeits. The results revealed that this HPLC-DAD-ESI-MS/MS analytical method established could be adopted as the comprehensive quality control method of MF.