A reverse phase high performance liquid chromatographic method was developed for estimation of Gentamycin sulfate in poultry meat. Samples were prepared by an extraction with phosphate buffer/TCA followed by a solid phase cleanup and then UV detector. The separation was achieved by C18 (4.6 mm i.d., 250 mm, 5 µm, Agilent Co.) column and the isocratic mobile phase of (0.1M) of Triflouroacetic acid: methanol (92:8v/v) was used, at a column temperature of 24 C at a flow rate of 1.5 ml/min and injection volume 50µl. Detection was carried out at 280 nm. Retention time of Gentamycin sulfate was found to be approximately 6 min. and analytical conditions gave recoveries in the range of 98.9% to 103.3% at spike levels of (50-1000 µg/kg), and relative standard deviations (RSDs) lower than 1% in all the cases. The repeatability and reproducibility results were in range of 2-10% and 3-15% respectively. Additionally, the limits of detection (LOD) (25µg/kg) and the limits of quantification (LOQ) (50 µg/kg) the method has been validated for linearity, accuracy and precision. Linearity for Gentamycin was in the range of( 50-1000 µg/kg),. The percentage recoveries obtained for Gentamycin sulfate found to be in range of 101%. Developed method was found to be accurate, precise, selective and rapid for simultaneous estimation of Gentamycin sulfate residues in poultry muscles that all standards in this method were added on poultry muscles to prepare a spiked sample for levels (50-1000µg/kg).