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Withania is a small shrub possessing medicinal value commonly found in the Indian subcontinent. Quantitative determination of withaferin-A, was undertaken to provide an easy and simple analytical method, which can be used as a routine quality control method. RP-HPLC was performed using acetonitrile and water as mobile phase. The detection and quantification was performed at a wavelength of 225 nm. Linearity of detector response for withaferin-A was between the concentrations 0.005% to 0.02%. The correlation coefficient obtained for the linearity was 0.993. The recovery value of standard withaferin-A was 94.4%. Low value of standard deviation and coefficient of variation, are indicative of high precision of the method. The system was successfully used to investigate the marker compound in different plant parts of W. coagulans and W. somnifera.