The invention discloses a method for determining active ingredients of a rhizoma smilacis glabrae medicinal material, relating to the filed of medicament analysis. In the invention, a fingerprint taking an astilbin chromatographic peak as an internal reference peak is established. The method comprises the following steps of: (a) preparing a test article solution; (b) taking astilbin as a reference substance and taking 0.10mg/mL astilbin solution prepared from methanol as a reference substance solution; (c) chromatographic conditions: taking a chromatographic column made of octadecylsilane chemically bonded silica as filler; adopting gradient elution, detecting the wavelength of 325+/-2 nm and the column temperature of 30+/-5DEG C; calculating the theoretical column plate number not less than 3,000 according to astilbin peak; and (d) determining. The fingerprint established by rhizoma smilacis glabrae ethanol-soluble extractives contains active ingredients with main activity of rhizoma smilacis glabrae. The fingerprint is used for judging the quality of rhizoma smilacis glabrae and the one sidedness for judging the quality of rhizoma smilacis glabrae by determining one or two chemical components can be avoided. The method has the advantages of simpleness, convenience, stability, high precision, favorable reproduction, easy grasp and capability of rapidly and accurately identifying the authenticity and merits of products.