Objectives: The aim of this study was to develop and optimize a simple, cost-effective, and robust high-performance liquid chromatography (HPLC) method by taking an experimental design approach to the assay and dissolution analysis of amlodipine besylate and enalapril maleate from a fixed-dose combination tablet. Materials and Methods: The chromatographic analysis was performed on a C18 column (4.6x250 mm id., particle size of 5 μm). The injection volume was 5 μL, and the detection wavelength was 215 nm. A Box-Behnken design was used to test the robustness of the method. The flow rate (1, 1.2, and 1.4 mL/min), column temperature (25°C, 30°C, and 35°C), methanol ratio of the mobile phase (5, 10, and 15%), and pH of the mobile phase (2.8, 3, and 3.2) were selected as independent variables. The method was validated according to International Conference on Harmonization guidelines. Dissolution of the tablets was performed by using USP apparatus 2 and analyzed using the optimized HPLC method. Multivariate linear regression analysis and ANOVA were used in the statistical evaluation. Results: Linear models were fitted for all variables. The flow rate was the most significant factor affecting the APIs’ concentrations. The optimized method included the following parameters: Column temperature of 25°C, 10% methanol as the mobile phase, pH of 2.95, and flow rate of 1.205 mL/min. Retention times were 3.8 min and 7.9 min for enalapril and amlodipine, respectively. The method was found to be linear in the range of 0.8-24 μg/mL (R2 >0.999) and 1.6-48 μg/mL (R2 >0.999) for amlodipine and enalapril, respectively. Both APIs were dissolved more than 85% within 10 min. Conclusion: The experimental design was proved as a useful tool for the determination and separation of enalapril maleate and amlodipine besylate in dosage forms. The optimized method can be used for in vitro performance and quality control tests of fixed-dose tablet combinations containing enalapril maleate and amlodipine besylate.