A novel approach was used to develop and validate a rapid, accurate, precise, simple, efficient and reproducible isocratic Reversed Phase-High Performance Liquid Chromatographic (RP-HPLC-DAD) method for the estimation of Lenvatinib Mesylate in bulk and pharmaceutical dosage form. Lenvatinib Mesylate were separated using Kromasil C18 column (250mm×4.6 mm, 5mm particle size), Waters Alliance e2695 HPLC system with 2998 PDA detector and the mobile phase contained a mixture of 0.01M Ammonium acetate (pH adjusted to 3.5 with orthophosphoric acid) and Methanol (30:70, v/v). The flow rate was set to 1ml/min with the responses measured at 309nm. The retention time of Lenvatinib Mesylate was found to be 3.733min.Linearity was established for Lenvatinib Mesylate in the range of 10-125μg/ml with correlation coefficient (r2=0.999). The percentage recoveries were between 100.3% to 100.6%. Validation parameters such as specificity, linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantitation (LOQ) were evaluated for the method according to the International Conference on Harmonization (ICH) Q2 R1 guidelines. The developed method was successfully applied for the quantification and hyphenated instrumental analysis.