Sensitive, simple, and accurate high-performance thin layer chromatographic method has been established for determination of β-sitosterol and Lupeol both simultaneously in Artocarpus lakoocha Roxb. leaf powder. The chromatographic separation was performed on silica gel 60 F254 HPTLC plate, with Toluene: Methanol: Formic acid, 7.0:2.0:0.3 (v/v/v), as mobile phase. After development, plates were treated with Methanolic Sulphuric acid Reagent. Detection and quantification were performed by densitometry at 366 nm in fluorescence mode. The developed method was then validated using statistical analysis. This will help in qualitative analysis of plant material using fingerprint pattern and quantitative analysis of β-sitosterol and Lupeol.