A new, rapid, accurate, and precise reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous quantification of gymnemagenin and 18β-glycyrrhetinic acid in herbal drug formulation has been developed and validated. To obtain gymnemagenin, polyherbal tablet formulation was subjected to acid hydrolysis followed by base hydrolysis and extraction with ethyl acetate. 18β-Glycyrrhetinic acid was obtained after acid hydrolysis of polyherbal formulation followed by extraction with chloroform. The chromatographic separation was achieved on Thermo Synchronis C18 analytical column (250 × 4.6 mm i.d., 5 μm) at 218 nm wavelength. The mobile phase comprising of methanol: water, pH 2.8, adjusted with orthophosphoric acid (92:08, v/v). The flow rate was set to 0.8 mLmin-1. The retention time for gymnemagenin and 18β-glycyrrhetinic acid was found to be 3.82 and 7.15 min, respectively. Validation of the HPLC method was carried out as per International Conference on Harmonization (ICH) Q2 (R1) guidelines. The calibration curve was found to be linear over a range of 50 - 1000 μgmL-1 for gymnemagenin and 50 - 500 μgmL-1 for 18β-glycyrrhetinic acid. The method has been applied for the analysis of marketed formulation. The content of gymnemagenin and 18β-glycyrrhetinic acid was found to be 0.2040 and 0.7431 %, respectively.