A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method for determination of S (-) Amlodipine Besylate (SAB) and its related substance i.e. R (+) Amlodipine Besylate (RAB) in tablet formulation was developed and validated. The isomer separation was carried out using simple isocratic reversed-phase HPLC method using a chiral column. Isocratic elution at a flow rate of 1.0 mL/min was employed on chiral column Ultron- ES-OVM, (250 x 4.6 mm, 5 μm). The mobile phase consisted of (Buffer: ACN) (78:22, v/v) in which buffer is mixture of 0.01M disodium hydrogen phosphate dihydrate and 0.01 M potassium dihydrogen phosphate. The UV detection wavelength was 237 nm and 20 μL of sample was injected. Method was validated with respect to specificity, linearity, precision accuracy and robustness. The method was found to be linear for the concentration range of 0.04 – 0.06 mg/mL of SAB with correlation coefficient r = 0.9998. The proposed method was applied successfully for quantification of SAB and RAB in tablet formulation. The method was found to be simple, accurate, sensitive and reproducible. It can be successfully utilized for routine analysis in quality control laboratory.