A simple, rapid, quantitative, and validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous estimation of quercetin (QU), rutin (RU), and gallic acid (GA) in the methanolic extract of Amaranthus tricolor L. aerial parts. Densitometric analysis of QU, RU, and GA were carried out in the absorbance mode at 254 nm. The method gave spot at RF 0.49, 0.14, and 0.28 corresponding to quercetin, rutin, and gallic acid, respectively. The limit of detection (22.31, 14.12, and 16.24 ng per spot) and limit of quantification (67.80, 55.32, and 52.54 ng per spot), respectively, were confirmed with the mobile phase toluene—ethyl acetate—formic acid in a ratio of 7:5:1 (v/v). Linear regression analysis data for the calibration plot for QU, RU, and GA showed a good linear relationship with a correlation coefficient r > 0.9955 in the concentration range of 200–800 ng per spot, 200–500 ng per spot, and 200–600 ng per spot, respectively. The method was validated for linearity, accuracy, precision, detection, and quantification limits, specificity, and robustness as per the International Conference on Harmonization (ICH) guidelines. The proposed validated high-performance thin-layer chromatographic method provides a novel approach for the quality control and standardization of A. tricolor L.
