An accurate, simple, rapid and sensitive HPLC method has been developed for the determination of mebhydroline napadisylate in the tablet. The Chromatography was performed on a reversed phase C-18 column(150 mm × 4.6 mm id, 5μm) by isocratic elution, using a mobile phase of acetonitrile : ammonia 25% (80 : 20 v/v) at ambient temperature 25±5 °C and UV detection operates at 320 nm in an overall analysis time of about 10 min.The total retention time was 1.612 min with a flow rate of 1.0 ml/min. % 0f RSD values for precision is found to be 0.293 (< 2).The limits of detection (LOD) and quantification (LOQ) were 0.03µg/ml and 0.096133µg/ml, respectively. The correlation coefficient for Mebhydroline Napadisylate 0.9972 indicates linearity of the methods within the limits. The linear range of determination for Mebhydroline Napadisylate was 100-500μg/ml. However, the change in flow rate and column temperature also did not show any significant variance. The % recovery was found to be 99.70%-99.41%.  As per ICH guidelines the proposed method is fully validated and found to be linear over a workable drug concentration, accurate, precise and robust.  This HPLC method is selective, precise, and accurate and can be used for routine analysis of pharmaceutical preparation in industrial quality-control laboratories.