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A high-performance thin-layer chromatographic (HPTLC) method was developed and validated as per ICH (International Conferences on Harmonization) guidelines for simultaneous quantification of three steviol glycosides, i.e. steviolbioside, stevioside and rebaudioside-A in Stevia rebaudiana leaves. For achieving good separation, mobile phase of ethyl acetate–ethanol–water (80:20:12, v/v/v) on pre-coated silica gel 60 F₂₅₄ HPTLC plates were used. The densitometric quantification of steviol glycosides was carried out at λ=510nm in reflection–absorption mode after spraying with acetic anhydride:sulphuric acid:ethanol reagent. The calibration curves were linear in the range of 160–960ng/spot for steviolbioside, 1–6μg/spot for stevioside and 0.5–3μg/spot for rebaudioside-A with good correlation coefficients (0.998–0.999). The method was found to be reproducible for quantitative analysis of steviol glycosides in …