A simple and reproducible UV- spectrophotometric method for the quantitative determination of Glycyrrhetinic acid in Pratishyayghna kwath (PK) were developed and validated in the present work. The parameters linearity, precision, accuracy, and standard error were studies according to Indian herbal pharmacopiea. In this present study a new, simple, rapid, sensitive, precise and economic spectrophotometric method in ultraviolet region has been developed for the determination of Glycyrrhetinic acid in laboratory herbal formulation of Pratishyayghna kwath. Each ingredient was purchased from the local market and they were evaluated as per Indian herbal Pharmacopoeia and WHO guidelines. The % w/w content of Glycyrrhetinic acid in the laboratory preparations of Pratishyayghna kwath PK-I, PK-II, PK-III were found to be 1.27±0.03 %, 1.29±0.02 %1.25±0.04 % respectively. Glycyrrhetinic acid has the maximum wavelength at 204 nm and hence the UV spectrophotometric method was performed at 204 nm. The samples were prepared in methanol and method obeys Beers law in concentration ranges 10- 50μg/ml. The content of Glycyrrhetinic acid in ayurvedic formulation was determined. The results of analysis have been validated statistically and confirmed the accuracy of the proposed method. Hence the proposed method can be used for the reliable quantification of Glycyrrhetinic acid in herbal formulation.