This research manuscript describes simple, sensitive, accurate, precise and repeatable reverse phase high performance liquid chromatography method for the estimation of Chlorhexidine gluconate in mouthwash. The sample was analyzed by reverse phase ACE 5 C18 column (150 mm × 4.6 mm i.d, 5 μm particle size) as stationary phase; acetonitrile : methanol : triethyl Amine (0.1 %) PH 3.0 (22: 49: 29, v/v/v) as a mobile phase at a flow rate of 0.8 ml/min. Quantification was achieved with Photo Diode Array detector at 258 nm. The retention time for chlorhexidine gluconate was found to be 2.477 min. The linearity was obtained in the concentration range of 10 -80 µg/ml for chlorhexidine gluconate. The method was successfully applied to mouthwash because no chromatographic interferences from formulation excipients were found. The method retained its accuracy and precision when the standard addition technique was applied.