Herbal drugs need to be standardized for their worldwide acceptance as it ensures the therapeutic efficacy and safety of the drugs. The phytopharmaceutical guidelines of the Central Drugs Standards Control Organization for botanical products suggest selecting a variety of compounds, i.e., a minimum of four phytoconstituents as the index for quality control of these products. Hence, for the quality control of multicomponent herbal formulations, simultaneous estimation of phytoconstituents would be a wise choice. The present work aimed to develop a simple and precise high-performance thin-layer chromatographic (HPTLC) method for the simultaneous estimation of berberine, mangiferin, gallic acid, and quercetin in Amritamehari churnam. Separation of phytoconstituents was carried out on silica gel 60F254 plates via a linear ascending technique using toluene‒ethyl acetate‒formic acid‒methanol (5:4:1:1, V/V) as the mobile phase. Densitometric scanning was performed at 254 nm to quantify the spots. The RF values of mangiferin, berberine, gallic acid, and quercetin were found to be 0.134, 0.325, 0.493, and 0.643, respectively. The proposed HPTLC method was validated as per the International Council for Harmonisation guidelines for linearity, accuracy, precision, limits of detection and quantification, robustness, and specificity. The calibration curves were linear with the correlation coefficients 0.99617, 0.99529, 0.99741, and 0.99845 for mangiferin, berberine, gallic acid, and quercetin, respectively. The developed method was successfully applied for the simultaneous determination of berberine, mangiferin, gallic acid, and quercetin in the commercial polyherbal formulation. The present HPTLC method, which can be used for the standardization of Amritamehari churnam, is being reported for the first time with these four phytoconstituents.