The roots of Vanda roxburghii R.Br. (family Orchidaceae), known as ‘Rasna’ in the Ayurvedic system of medicine, are used to treat a variety of ailments, including rheumatism, dyspepsia, bronchitis, inflammations, diseases of the abdomen, hiccough and tremors and as an antipyretic agent. Chemically, V. roxburghii R.Br. has been reported to contain β-sitosterol, γ-sitosterol, heptacosane, octacosanol, acetyl tetracosylferulate, 17-β-hydroxy-14,20-epoxy-1- oxo-[22R]-3β-[O-β-D-glucopyranosyl]-5,24-withadienolide and melianin. Sterols are anti-inflammatory agents. β- sitosterol has been shown to possess anti-inflammatory and anti-pyretic properties. In this context, a method for quantitative estimation of β-sitosterol in the methanolic extract of the roots of Vanda roxburghii R.Br. has been developed. The HPTLC method used for the standardization was validated for the parameters like specificity, limits of detection and quantification, linearity, precision, accuracy and recovery. The content of β-sitosterol in the methanolic extract was found to be 0.016 %w/w.