Hydrolysis of stevioside (I) with crude hesperidinase afforded its genuine aglycone, steviol (III) quantitatively. Rebaudioside-A, which was newly isolated from Stevia rebaudiana along with I, yielded isosteviol (II) on acid hydrolysis, was hydrolyzed partially, and did not give III with this crude enzyme. The optimal condition for the enzymic hydrolysis of I and quantitative analysis of the resulting III by gas chromatography were studied to establish the selective determination of I in Stevia leaves. This procedure can be applied to the simple determination of I in orange juice without any pretreatment.