Abstract: Bulbus Fritillaria (BF), one of the derivatives from the plant of various Fritillaria species, has been commonly used as expectorant and antitussive herb in traditional Chinese medicine. The value of BF is relative related to its quality, however, there is no reliable method to evaluate its quality. Therefore, it is necessary to develop a rapid and reliable method for classification of species of BF. In this study, the QuEChERS (quick, easy, cheap, effective, rugged, and safe) as sample preparation method and combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was proposed to determine trace alkaloids of BF. The main bioactive isosteroidal alkaloids analyzed are peimine, peiminine and peimisine. The extraction efficiency of QuEChERS were systematically studied and the optimal extraction conditions were as follows: extraction of three alkaloids by using 3 mL extraction solution of ethyl acetate-water (75∶25, V/V) which is containing 3% ammonium hydroxide and 1 g of ammonium carbonate, and then mixing by vortex condition for 5 min. The extract was cleaned with 10 mg of anhydrous magnesium sulfate and 20 mg of florisil. The extracted isosteroidal alkaloids were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The quantification was performed by extracted ion chromatography and MS/MS spectra was applied for identification of isosteroidal alkaloids. The linear range for peimisine is from 0.1 to 20 μg/g and for peiminine and peimine are from 0.05 to 10 μg/g with coefficients of determination (R²) above 0.992 3. The limit of detection (LODs) of the proposed method range from 0.005 to 0.03 μg/g. Intra-day and inter-day precisions represented in RSD% are in a range of 1.1%-17.5%. The recoveries are in a range of 89.6%-97.0%. The amount of peimisine, peiminine, peimine in a real Chuan-Beimu sample were detected by the proposed method and the amount are at 4.35, 0.92, 1.39 μg/g, respectively.