A rapid and precise reverse phase high-performance liquid chromatographic method has been developed for the validated of Lefamulin, in its pure form as well as in tablet dosage form. Chromatography was carried out on a Symmetry C18 (4.6 x 150mm, 5µm) column using a mixture of Methanol and water (45:55% v/v) as the mobile phase at a flow rate of 0.8ml/min, the detection was carried out at 260nm. The retention time of the Lefamulin was 2.359 ±0.02min respectively. The method produces linear responses in the concentration range of 24-120mg/ml of Lefamulin. The method precision for the determination of assay was below 2.0%RSD. The method is useful in the quality control of bulk and pharmaceutical formulations. The method was validated for accuracy, precision, linearity, robustness, ruggedness and LOD & LOQ of standard solution. The developed RP-HPLC method was found to be accurate, precise, linear, and robust and was successful applied to a pharmaceutical tablet formulation for qualitative estimation of Lefamulin in Bulk form and Marketed Pharmaceutical Dosage forms.