A simple, rapid, and specific thin layer chromatographic (TLC) method has been developed and validated for the simultaneous estimation of icariin and l-arginine from commercial polyherbal formulations for sexual dysfunction. The separation of the methanol extract of these formulations was achieved on silica gel 60 F254 aluminum backed TLC plates by using ethyl acetate-acetone-glacial acetic acid-formic acid-water 12:2:1:2:2 (υ/υ) as mobile phase. Densitometric analysis of icariin and l-arginine was monitored in absorbance mode at 270 and 195 nm, respectively. The linear regression analysis data for the calibration plots for icariin and l-arginine showed good linear relationship with r2 = 0.9984 +- 0.01 and 0.9968 +- 0.02, in the concentration ranges of 250–750 and 500–1500 ng/spot, respectively. The method was validated for precision, robustness, and recovery. The average percentage recovery was found to be 98.26% for icariin and 99.63% for l-arginine. The limits of detection and quantitation were 72, 116 and 238, 383 ng/spot, respectively, for icariin and l-arginine. Statistical analysis proves that the method is repeatable and selective for the estimation of the targeted drugs. Since the proposed mobile phase effectively resolves the icariin and l-arginine, this method can be applied for the identification and quantitation of these components in herbal extracts and marketed formulations.