AbstractMangiferin (polyphenolic xanthone) and scopoletin (phenolic coumarin) are well-studied biological markers present in Canscora decussata(Roxb.) Roem. & Schult., Gentianaceae. The objective set for the present studies is to establish and develop a new, simple, selective, sensitive, and precise high performance thin layer chromatography method for the simultaneous estimation of mangiferin and scopoletin in hydroalcoholic extract of C. decussata. The thin layer chromatographic separation of these biomarkers was carried out on aluminum plate pre-coated with silica gel 60F254, eluted with ethyl acetate:acetic acid:formic acid:water (10:0.5:0.5:1.5). The plate was then dried and densitometric scanning was performed at 254 nm using a Camag TLC scanner III. The system was found to give compact spots for mangiferin (RF 0.22) and scopoletin (RF 0.78). A good relationship of linear precision between the concentrations (100–600 ng/spot) and peak areas was obtained with correlation coefficient (r) of 0.9979 (mangiferin) and 0.9962 (scopoletin), respectively. The limits of detection and limit of quantification were determined to be 46 and 94 ng/spot for mangiferin and 31 and 78 ng/spot for scopoletin respectively. The percentage of recovery was found from 99.91 to 99.94% for mangiferin and 99.75 to 99.86% for scopoletin. Results obtained from recovery studies showed excellent reliability and reproducibility of the method. Present communication on validated high performance thin layer chromatography method may provide a new, selective, sensitive, and precise method to estimate mangiferin and scopoletin as phytomarkers in the hydroalcoholic extract of C. decussata used in Ayurvedic formulations.