Variability in phytochemical composition of herbal raw materials necessitates robust marker-based standardization to ensure quality, safety, and therapeutic consistency. Rutin, a bioactive flavonol glycoside, is widely used as a quality control marker in flavonoid-rich medicinal plants. The present study aimed to develop and apply a validated HPTLC method for the quantitative estimation of rutin in selected medicinal plant extracts commonly used in traditional systems of medicine. Methanolic extracts of Azadirachta indica (flowers and leaves), Gossypium arboreum (flowers), Cassia auriculata (bark), Pachygone brevifolia (leaves), Cuminum cyminum (fruits), and Vitex trifolia (leaves) were analyzed using HPTLC. Chromatographic separation was achieved on silica gel 60 F254 plates using toluene: ethyl acetate: formic acid: methanol (3:6:1.6:0.4, v/v/v/v) as the mobile phase. Densitometric scanning was performed at 254 nm.: Rutin was well resolved with Rf values ranging from 0.16 to 0.20. Quantitative analysis revealed rutin content of 0.28% (A. indica flowers), 0.22% (A. indica leaves), 0.32% (C. auriculata bark), 0.01% (G. arboreum flowers), 0.33% (P. brevifolia leaves), and 0.13% (V. trifolia leaves). The HPTLC fingerprints demonstrated good resolution and reproducibility. The developed HPTLC method is simple, rapid, and reliable for the quantification of rutin in medicinal plant extracts. The findings support the use of rutin as a suitable phytochemical marker for quality control and standardization of flavonoid-rich herbal raw materials and traditional polyherbal formulations.