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Flavonoids compounds have anti-oxidation properties in that they remove free radicals, reduce blood pressure and reduce vessel thrombosis. So many extraction methods are available, but the selection of specific solvent system for the separation and quantification of quercetin is important. It can be easily analyzed by chromatographic methods, but before testing a new type of plant material it is opportune to optimize chromatographic conditions. The aim of this study is to optimize the chromatographic conditions in TLC of flavonoids, as standard compounds that may be present in Euphorbia hirta. We compared 10 different mobile phases, to find the most appropriate mobile phase. The results showed that the optimal extraction parameters were as followings: concentration of methanol solution was 60%, with ethylacetate (40%).