Ocimum sanctum (family Lamiaceae) is a reputed drug of Ayurveda, commonly known as Tulasi. In the present work, we quantified 4 marker compounds, viz., eugenol, luteolin, ursolic acid, and oleanolic acid, from the leaf of green and black varieties of O. sanctum using high-performance thin-layer chromatography (HPTLC) with densitometry. The methods were found to be precise, with relative standard deviation (RSD) values for intraday analyses in the range of 0.52 to 0.91%, 0.77 to 1.29%, 0.11 to 0.16%, and 0.34 to 0.42% and for interday analyses in the range of 0.73 to 0.96%, 1.02 to 2.08%, 0.11 to 0.12%, and 0.39 to 0.64% for different concentrations of eugenol, luteolin, ursolic acid, and oleanolic acid, respectively. Instrumental RSD values were 0.24, 0.39, 0.21, and 0.18% for eugenol, luteolin, ursolic acid, and oleanolic acid, respectively. Accuracy of the methods was checked by conducting a recovery study at 3 different levels for the 4 compounds, and the average recoveries were found to be 99.73, 99.3, 100.58, and 100.57%, respectively. Eugenol content ranged from 0.175 to 0.362% (w/w) and luteolin from 0.019 to 0.046% (w/w) in the samples analyzed. Green variety was found to contain higher amounts of ursolic acid [0.478 and 0.348% (w/w), from Sources 1 and 2, respectively] than the black variety [0.252 and 0.264% (w/w) from Sources 1 and 2, respectively]. Black variety had 0.174 and 0.218% (w/w) of oleanolic acid from Sources 1 and 2, respectively, while it was not detected in the green variety. Ursolic acid and oleanolic acid ran at the same Rf value and could not be resolved in several solvent systems tried. However, we observed that only ursolic acid gave yellow fluorescence under 366 nm ultraviolet light after derivatization with anisaldehyde-sulfuric acid reagent. The HPTLC-densitometry methods for the quantification of the 4 markers in O. sanctum leaf will have the applicability in quality control.