Objective:This study aimed to develop and validate a method using a high-performance liquid chromatography (HPLC) to perform a quantitative analysis of nebivolol (NEB) and amlodipine (AML) along with some related substances in the synthetic mixture.Methods:The separation in the described chromatographic system was accomplished using a mobile phase consisting of a mixture of acetate buffer (pH: 4.5) and acetonitrile and a HPLC C18 column (150 mm x 4.6 mm, 2.6 μm) with gradient elution on a consistent flow rate of 1.3 mL/min. Photodiode array detection was carried out at a wavelength of 265 nm. According to The International Conference on Harmonisation guidelines, the drug was exposed to various stress conditions; including photolysis, oxidation, thermal degradation and hydrolysis under acidic, basic and neutral mediums.Results:Ranges of detection and quantitation limits were determined to be 0.2-10.0 μg.mL-1 and 0.25-10.0 μg.mL-1 for NEB and AML, respectively. The relative standard deviation values within and between days precision were determined to be