Aerial parts of Barleria prionitis Linn. (Acanthaceae) have shown anti-respiratory syncytial virus, anti-arthritic, anti-inflammatory, hepatoprotective, anti-stress, and immunorestorative activities. The two iridoid glycosides, shanzhiside methyl ester (SME) and barlerin, are the active principles of B. prionitis (BP). For the quality control of BP, rapid methods of quantitation are desirable. Therefore, a simple and reproducible high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantification of SME and barlerin in BP was validated. The marker compounds were isolated, purified, and authenticated by spectral analysis. Precoated silica gel 60 F 254 TLC plates were used as a stationary phase and chloroform-methanol in the proportion of 80:20 (v/v) as the mobile phase. The method was validated in terms of linearity, precision, robustness, accuracy, and limits of detection and quantification. Quantification was performed in the absorbance mode at a wavelength of 240 nm using Deuterium lamp. The linearity was found to be in the range of 200-1000 ng spot -1 for both SME and barlerin, with correlation coefficients of 0.9956 and 0.9932, respectively. The percent recoveries for SME and barlerin were in the range of 99.20 to 99.54%, and 98.93 to 99.19%, respectively. Limits of detection and quantification were found to be 12.95 ng and 21.688 ng, and 17.861 ng and 31.051 ng, respectively, for SME and barlerin. The content (% w/w) of SME and barlerin was found to be 4.91 and 4.69, respectively, in methanol extract of BP. Statistical analysis of the data showed that the proposed method is simple, sensitive, precise, robust and reproducible, specific, and can be employed profitably in place of HPLC.