A new, simple, sensitive, selective, precise and robust high-performance thin-layer chromatographic (HPTLC) method for analysis of glycyrrhizin has been developed and validated for the determination of glycyrrhizin in herbal extracts and in herbal gel. The analyte was extracted with ethanol and applied on TLC aluminium plates along with standard using Linomat 5 spray on sample applicator (CAMAG). Analysis of glycyrrhizin was performed on pre-coated TLC aluminium plates with silica gel as the stationary phase and prewashed with methanol. Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of ethyl acetate-methanol-water-formic acid (15: 2: 1: 1 v/v/v/v). Spectrodensitometric scanning was performed by TLC scanner III (CAMAG) in absorbance mode at the wavelength of 252nm. The system was found to give compact spots for glycyrrhizin (Rf value of 0.34±0.04). The linear regression analysis data for the calibration plots showed good linear relationship with r2= 0.9981 in the concentration range 2-15 µl with respect to peak area. According to the International Conference on Harmonization (ICH) guidelines the method was validated for precision, recovery, stability in solution and robustness. The glycyrrhizin content quantified from herbal extracts and from the formulation was found well within limits. Statistical analysis of the data showed that the method is reproducible and selective for the estimation of glycyrrhizin.