Background: Fructus Sophorae pill, one of the traditional Chinese medicine, was widely used for hemorrhoids, hypertension and odontalgia. This paper describes a sensitive and specific assay for the determination of the 15 active constituents (sophoricoside, genistin, genistein, rutin, quercetin, kaempferol, baicalein, baicalin, naringin, naringenin, hesperidin, neohesperidin, wogonin and cimifugin, prim-O-glucosylcimifugin) in Fructus Sophorae pill. Materials and Methods: Chromatographic separation was performed on a C 18 column with acidified aqueous methanol gradients at a flow rate of 0.8 mL/min. The identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was applied to quantification with switching electrospray ion source polarity between positive and negative modes. Results: The proposed method was used to analyze 40 batches of samples with good linearity (r, 0.9990-0.9999), intraday precisions (RSD, 0.14-2.55%), interday precisions (RSD, 0.51-2.81%), stability (RSD, 0.31-2.65%), and recovery (RSD, 1.29-2.95%) of the 15 compounds. In addition, the hierarchical cluster analysis, including a method called furthest neighbor and nearest neighbor, was employed to classify samples according to characteristics of the 15 constituents. Conclusion: The results indicated that the analytical method was rapid, reliable, simple and suitable for the quality evaluation of Fructus Sophorae pill.