Pharmacokinetics of gatifloxacin (Gatiquin® – Cipla Ltd. Ahmedabad., India) in six cross bred cow calves following intravenous (IV) administration (4 mg/kg body weight). Estimation of gatifloxacin in plasma samples was analyzed by microbiological assay technique by using E. coli as test organism. Kinetic parameters of gatifloxacin were calculated by using two-compartment open model. Therapeutic concentration of gatifloxacin (0.2 µg/ml) was maintained up to 16 h and the drug was detectable up to 24 h. Distribution half life (t1/2 α), elimination half life (t1/2 β), mean residential time (MRT), Volume of distribution during area under curve (Vdarea) and total body clearance (ClB) of 0.125 ± 0.01 h, 13.17 ± 0.16 h, 18.54 ± 0.26 h, 1.81 ± 0.02 L/kg and 0.076 ± 0.01 L/kg/h, respectively were obtained for gatifloxacin. For maintaining therapeutic concentration of 0.2 µg/mL, a loading dose (D*) of around 0.8 mg/kg and maintenance dose (D0) of 0.4 mg/kg may be used at the dosage interval (τ) of 16 h for treating systemic infections.