[Objective]To establish an HPLC method for the determination of the contents of stevioside and rebaudioside A in Stevia rebaudiana.[Methods]The separation was performed by HPLC method using NH2column(150 mm×4.6 mm,φ5μm),mobile phase of wateracetonitrile(17∶83)with linear elution and detection wavelength at 210 nm.[Results]The optimal method for isolating stevioside and rebaudioside A from stevia leaves was water-extraction at 60℃for 90 min.The linear ranges of stevioside and rebaudioside A in Stevia rebaudiana were determined as from 0.2 to 2 mg/mL.The concentration of stevioside was calculated according to formula:y=2×106x+27 581(R2=0.994 9);the concentration of rebaudioside A was calculated according to formula:RA:y=2×106x+38 395(R2=0.997 5).[Conclusion]This reliable and robust HPLC method proposed here can be applied for the quantification of steviol glycosides in Stevia rebaudiana leaves in laboratories and quality control in industrial production.