To develop rapid, rugged, precise and an accurate stability indicating analytical method for estimation of related substances in Ursodeoxycholic Acid tablets. The separation of impurities and Ursodeoxycholic Acid drug is achieved by an isocratic chromatographic method on C18, 250 mm x 4.6 mm, 5µm column. The mobile phase consists of buffer, acetonitrile and methanol in the ratio of 35:28:37 v/v/v pumped at a flow rate of 1.0 ml/minute 35:37:28v/v/v pumped at a flow rate of 1.0 ml/minute. The detection was carried out by using refractive index (RI) detector. The proposed chromatographic method was validated and found to be linear over the concentration range from LOQ to 150.0% of impurity limit level.  Overall mean recovery of Chenodeoxycholic acid impurity was found to be 100.6±4.0%w/w The method was found to be simple, stability indicating, precise, accurate and robust which can be utilized for estimation of related substances in Ursodeoxycholic Acid tablets