The present paper deals with HPLC method development and validation for determination of Arginine, Lysine and Histidine in drug substance and drug product without derivatisation by High Performance Liquid Chromatography (HPLC) using HILIC column technique. The above amino acids are eluted by using isocratic mobile phase which contains solution A as 50 mM Potassium dihydrogen phosphate and solution B as Acetonitrile in the ratio of 70:30. The Limit of Quantitation (LOQ) of Arginine, Lysine and Histidine is 0.22 μg/mL, 6.7 μg/mL and 0.14 μg/mL respectively. The Limit of Detection (LOD) of Arginine, Lysine and Histidine is 0.066 μg/mL, 2.01 μg/mL and 0.042 μg/mL respectively. The method is accurate, linear and precise from LOQ level to 150% level with respect to analyte concentration for the analysis of Arginine, Lysine and Histidine in bulk drug substance. The proposed method is accurate to analyse the Arginine, Lysine and Histidine in drug product at 50% level to 150% level with respect to analyte concentration. The method is specific to resolve the degradation impurity formed under Acidic, Basic and Oxidative condition.