Abstract: Flavonoids and the other phenolic compounds are commonly known as plant secondary metabolites that hold an aromatic ring bearing at least one hydroxyl groups. Flavonoids are principal active constituents have been used to treatment of various human diseases. The plant Desmodium gangeticum (DC), Family-Fabaceae has been used in folklore medicine in the treatment of various ailments. Many of the Ayurvedic formulations contain this medicinal plant and considered as a Master of Medicinal Plant in Ayurveda due to its wide uses in formulations. A medicinal benefit includes bitter tonic, febrifuge, digestive, anti-emetic, antipyretic & anti-inflammatory activity. The chromatographic separation was achieved by using a C-18 column with dimension of 4.6 mm I.D.X 250 mm and particle size of 5μm. The mobile phase contain methanol: water (70:30). The flow rate was 0 .5 mL/min, and a column temperature of 25°C. The injection volume was 25μl, and UV detection was achieved at 254 nm. Effective separation and quantification was achieved in less than 10 min. The method was simple, accurate, precise and could be successfully applied for the characterization of flavonoids in aqueous extract of DC.