Objective：To establish methods of high performance liquid chromatograph(HPLC)so as to determine content of rubrofusarin gentiobioside，assiaside C and Aurantio-obtusifolin-6-O-β-D-glucopyranoside of cassia seed.Methods：Performed Zorbax Eclipse XDB-C18 column at the temperature of 30℃，and used acetonitrile and tetrahydrofuran(A)-1%acetic acid(B)(A∶B=30∶70)as mobile phase by isocratic elution，with the flow rate being 1.0 mL/min and detection wavelength at 278 nm.Results：Three effective components achieved a baseline resolution.Rubrofusarin gentiobioside，assiaside C and Aurantio-obtusifolin-6-O-β-D-glucopyranoside respectively demonstrated a good linear relationship with the peak area in the range of 0.136 4～1.091 μg，0.117 9～0.943 μg，and 0.126 3～1.010 μg.The respective typical equation was Y=3.78×106X-143 92，Y=2.67×106X-784 35，Y=3.15×106X-108 435 respectively，and their average recovery was 100.53%，98.95%and 102.30% respectively.For the determination of cassia seed from different habitats，the result indicated there was great difference among the content of these three effective components.Conclusion：By methodologyverification，the determination method is accurate，reliable，and of good reproducibility.It can be an effective reference to the evaluation and quality control of cassia seed.