Estimation of related substances by using high-performance liquid chromatographic method was developed and validated for the determination of Flavoxate Hydrochloride. The method is simple, highly sensitive, and selective and is capable of quantitative determination of Flavoxate Hydrochloride. The chromatographic separation is achieved by injecting 20μL standard solution of Flavoxate Hydrochloride into HPLC system with PDA detector using a Hypersil ODS, 5μm (150 x4.6) mm column. The mobile phase consists of pH 2.75 buffer and Acetonitrile. The flow rate was set at 1.0 ml/min with column and sampler temperatures at 25°C and ambient respectively and runtime was optimized to 25 min. The developed LC method was validated with respect to specificity, precision, linearity, ruggedness, stability of analytical solution and robustness.