This document describes the development and validation of a reverse phase high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of amitriptyline and chlordiazepoxide. The method utilizes a C18 column, mobile phase of phosphate buffer and acetonitrile (55:45), flow rate of 1 ml/min, and detection wavelength of 252 nm. Linearity was achieved between 25-150 μg/ml for amitriptyline and 10-60 μg/ml for chlordiazepoxide. The method was validated for accuracy, precision, specificity, linearity, robustness and sensitivity. The developed RP-HPLC method allows for the simultaneous quantitative analysis of amitriptyline