Extensive research on Ficus species has shown their excellent cytotoxic potential which motivated the authors for further evaluation of its other species. In this article, the β-sitosterol content in the chloroform extract of the leaves of five Ficus species (Ficus carica [FCCE], Ficus nitida [FNCE], Ficus ingens [FICE], Ficus palmata [FPCE], and Ficus vasta [FVCE]) was estimated by a validated high-performance thin-layer chromatography (HPTLC) method along with cytotoxic activity. The chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with hexane and ethyl acetate (8:2, v/v) as the mobile phase. The developed plate was derivatized with p-anisaldehyde reagent, scanned, and quantified at λ = 550 nm. It furnished a compact and intense peak of β-sitosterol at RF = 0.17 ± 0.001. The contents of β-sitosterol (μg mg−1 of the dried weight of the extract) in the selected Ficus species were found as: FCCE (1.047 μg mg−1) > FVCE (0.771 μg mg−1) > FNCE (0.372 μg mg−1) > FPCE (0.309 μg mg−1)...