Gatifloxacin is a fluoroquinolone having broad-spectrum activity and good antibacterial activity at low plasma/tissue concentration. The present study was designed to investigate safety of gatifloxacin (10 mg/kg body weight) after repeated oral administration at 12 hours interval for 14 days in broiler chickens and to determine tissue concentration of the drug following oral administration (10 mg/kg body weight) for 5 days. Repeated oral administration of gatifloxacin in broiler chickens was found safe based on evaluation of hematological (Hb, PCV and TLC), biochemical (AST, ALT, ALP, LDH, Serum uric acid, Serum Creatinine, Blood glucose and Total bilirubin) and histopathology of liver, kidney, heart and joint cartilage. Drug concentration in tissue was determined using High Performance Liquid Chromatography (HPLC). The concentration of gatifloxacin was found 0.75 ± 0.04 µg/g after fourth dose and 0.22 ± 0.07 µg/g after tenth dose respectively in liver, whereas in skeletal muscles the concentration of gatifloxacin was below the limit of quantification after fourth dose and after tenth dose gatifloxacin was not detected.