One hundred one day day old broiler chickens were fed balanced rations ad libitum with free access to water. At 3 weeks in this study. 10 birds as a random samples were scarified for postmortem and bacteriological examination to detect Pasteurella and to serum plate agglutination test (SPA) to prove that free from Mycoplasma gallisipticum. Other birds were randomly divided into three groups of 30 chickens each. The first group was a control. The second and third groups administered tilmicosin in drinking water at concentrations of 30 and 60 mg/kg bw for 5 days. Six chickens of each group, three males and three females, were slaughtered on days 3, 7, 14 and 21 days after dosing. Theresults reveled significant increase in lymphocytes transformation test (LTT) in high dose administration of tilimicosin at the end of experiment. High dose of tilmicosin (H1,H2) caused decrease albumin levels and ɣ globulin. The result revealed no significant changes in serum creatinine in all period of experiment. Alanine amino transferrase (ALT) showed significant increase in groups treated with low and high dose in all period of experiment. Aspartate amino transeferrase (AST) showed non significant changes on group treated with low dose but high dose treated group revealed significant increase in 1^st and 2^nd periods only and return to normal level in the 3^rd period. Serum alkaline phosphatase (ALP) revealed significant increase in group treated with high dose in all period of experiment. The residue levels in both doses were significantly higher in liver than kidney or muscle at 3, 7, 14 day post treatment and not detected at 21 day post treatment in all examined samples including liver. The results indicated that tilmicosin residue was eliminated from muscle very quickly, but from liver very slowly. The minimum withdrawal time of 7 days for low dose and 14 days for high dose. The chromatograms obtained with this method was validated with limit of tilmicosin detection 10µg/kg.