||A new gradient reverse phase high performance liquid chromatography method has been developed for quantitative determination of related substances of Lysine Clonixinate (LC) and Cyclobenzaprine Hydrochloride (CBP) in pharmaceutical dosage forms. Chromatographic separation achieved on a column L1, C18 250 mm x 4.6 mm; 5 µm with 0.025M Potassium dihydrogen phosphate pH 3.0 buffer as mobile phase-A and Acetonitrile as mobile phase–B at a flow rate of 1.7 mL min-1. Diluent as water and acetonitrile of ratio 50:50 v/v with detection at 245 nm. The injection volume was 20 µL and a gradient program with run time of 60 minutes. The developed method validated according to ICH guideline with the parameters specificity, forced degradation (with stress conditions of acid, base, oxidative hydrolysis, thermal and photolytic degradation), limit of detection and limit of quantification, linearity, precision, intermediate precision, accuracy, robustness and ruggedness. From all validation parameters results the method proved as specific, stability indicating, precise, accurate, robust and rugged method.