The present paper proposes a method that includes optimization of extraction process and high-performance thin-layer chromatographic (HPTLC) method for the rapid and simple quantification of two isomeric anticancer compounds (isogarcinol and garcinol) in Garcinia indica fruit and its validation according to the International Conference on Harmonization (ICH) guidelines. HPTLC of isogarcinol and garcinol was performed on precoated silica 60 F254S HPTLC plates. For achieving good separation, the mobile phase of n-pentane-ethyl acetate-formic acid (7:3:0.5, v/v/v) was used. Densitometric determinations of the investigated compounds were carried out at 327 nm in reflection-absorption mode. The calibration curves were linear in the range of 990 to 6900 ng for isogarcinol and 200 to 1400 ng for garcinol. The method was found to be reproducible and convenient for quantitative analysis of garcinol and isogarcinol in different solvent extracts of G. indica fruit. Our work demonstrated the optimization of the extraction procedure — rapid and sensitive quantification of two isomeric anticancer compounds — and its validation in the fruit extract of G. indica.