High performance liquid chromatographic and 1st derivative spectrophotometric methods for simultaneous determination of chlorhexidine gluconate (CG) and benzydamine hydrochloride (BH) in gargle and spray preparations used for the treatment of dental illnesses, mouth and faucet infections and providing oral hygiene were developed and validated. CG and BH responses were respectively measured at 271 nm and 324 nm in 1st derivative spectrophotometry. In HPLC; the analysis of CG and BH together with internal standard hydrochlorothiazide (HCT) were performed using an analytical column of Nucleosil 100-5 C18 (5 μm, 250 × 4,6 mm i.d.). Substances were eluted by a mobile phase consisting of 40 mM triethylamine containing phosphate buffer (10 mM, pH 3.0) and acetonitrile (65:35, v/v) at a flow rate of 1 mL min-1. UV detector was set to 230 nm. Under these chromatographic conditions, retention times of HCT, CG and BH were 3.44, 5.42 and 8.58 min, respectively. Developed methods were validated for the parameters of stability, linearity, sensitivity, accuracy, precision, specificity, ruggedness and robustness given in current ICH Guideline. Linearity ranges for two substances were 1-60 μg mL-1 in HPLC and 1-50 μg mL-1 in 1st derivative spectrophotometry. Developed methods were accurate, precise, specific, sensitive, rugged and robust due to the outcome from validation study results. These methods were applied to the binary mixture preparations of CG and BH (gargle and spray) and their reliability for the analysis of these preparations are presented.