Betulinic acid (BA), a potent anticancer agent, occurs in more than 50 plant species worldwide, out of which Dillenia and Ziziphus species are found in Gujarat, India. The presented research work was aimed at the development of a single validated high-performance thin-layer chromatographic (HPTLC) method which can quantify BA from different plant species and thereof from different chemical environments. The analysis was performed on a thin-layer chromatographic (TLC) precoated silica gel 60 F254 plate using a mobile phase of toluene-chloroform-ethanol (4:4:1, v/v/v) and after derivatization with anisaldehyde sulfuric acid reagent showed magenta color spot at RF 0.71. Densitometric evaluation of BA was carried out at 525 nm. The suitability of this HPTLC method for quantitative determination of BA was proved by validation in accordance with the requirements of the International Conference on Harmonization (ICH) guidelines. The occurrence of BA is more frequent in bark or stem as compared to leaves. The content of BA was found to be the highest, 0.43%, in Dillenia indica bark.