A simple liquid chromatographic method was developed for the determination of gymnemagenin in leaves of a renowned anti-diabetic herb, Gymnema sylvestre. Gymnemagenin was obtained after acidic hydrolysis followed by basic hydrolysis of the sample and extraction into ethyl acetate. Analyte separation and quantitation were achieved by isocratic reversed-phase liquid chromatography and UV detection at 220 nm. The method involves the use of an RP-18e Lichrocart reversed-phase column (5 μm, 75 x 4 mm id) and a binary isocratic mobile-phase profile. Linearity was observed in the range of 9.18 to 720 μg mL-1 with correlation coefficient of 0.998. Relative standard deviation of linearity of the method was found to be 0.015%.Detection limit was 5.5 μg mL-1 and quantitation limit was 7.5 μg mL-1. Average recovery of 99.2 ± 0.54,
was obtained by spiking pre-analyzed samples with standard solution at 3 different concentration levels (80, 100, and 120%). Three leaf samples of G. sylvestre from three different regions, one marketed G. sylvestre extract and an anti-diabetic polyherbal formulation containing G. sylvestre leaf powder were analyzed by this method. They were found to contain 0.30-0.34. 5.9 and 0.125% w/w gymnemagenin respectively. The new method is comparatively simpler, reproducible and sensitive than the other reported methods.