In the present investigation, a simple, sensitive, precise and accurate Reverse phase high performance liquid Chromatography method developed and validated for estimation of Netilmicin in injection formulation. This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps. Netilmicin was freely soluble in ethanol, methanol and sparingly soluble in water. ACN: Methanol: water (10:30:60) was chosen as the mobile phase. The solvent system used in this method was economical. The %RSD values were within 2 and the method was found to be precise. The results expressed in tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods. This method can be used for the routine determination of Netilmicin in injection formulation and in other pharmaceutical dosage forms. Key words: RP-HPLC, Netlimicin, injection.