The present study was carried out to evaluate the withdrawal period of levofloxacin after repeated oral administration for 28 days in broiler chicken. The tissue samples of which were used for the standardization of the instrument, High performance liquid chromatography (HPLC). Group- T1 (n=30) birds received levofloxacin at 10 mg per kg body weight and Group- T2 (n=30) 20 mg per kg body weight orally for 28 days. While the control group (untreated group) given plane drinking water. For residues analysis and withdrawal time calculation, birds were kept under observation for two week prior to commencement of experiment and were provided antibiotic free standard broiler ration for fourteen days. The animal house was maintained at room temperature. Samples of liver and breast muscle were collected and the tissue samples were stored at 45°C. The drug concentration in tissue samples was determined by HPLC assay. The residue concentration of levofloxacin were gradually decreased in chicken liver and breast muscle samples starting from day one to day ten after the last dose subject to clinical examination in order to exclude the possibilities of disease. The broiler birds, Levofloxacin was estimated to have pre slaughter withdrawal period of four to five days in comparison with maximum residue level of 200, and 100 μg / kg for liver and breast muscle respectively. It was observed that slower elimination of levofloxacin from the body could be attributed to its lipophilicity and high tissue perfusion rate. The main objectives of present investigations are to study the residual effect of Levofloxacin in liver and breast muscles of broiler birds.