Rasayana churna is a well known rejuvenating formulation of Ayurveda, consisting of guduchi (Tinospora cordifolia), gokhru (Tribulus terrestris) and amla (Embelica officinalis). A UV- spectrophotometric method for the quantitative determination of berberine in Rasayana churna was developed and validated. The method involved measurement of absorbance of sample solutions at the absorption maxima of berberine (348 nm). The interference from other compounds was eliminated by processing the crude drug and the formulations to isolate alkaloidal fraction from them. The method was validated by determining linearity, precision and accuracy as per the ICH guidelines. Berberine obeyed Beer’s law in the concentration range of 2-20 μg/ml. The method proved to be quite accurate with a mean percentage recovery of 99.21%. Thus, the proposed method is simple, rapid, precise, accurate, and suitable for quantification of berberine content in samples of Rasayana churna.