This paper describes a method for determining enrofloxacin (ERFX) and it's main metabolite ciprofloxacin(CPFX), in animal plasma by RP-IPLC simultaneously. Dichloromethane was utilized as an extracting agent of plasma. Chromatographic conditions were ODS column with methanol-25 mmol/L tetrabutylammonium hydroxide (20:80, V/V, pH 3.0) as mobile phase, pipemidic acid as the internal standard and UV detection at 272 nm. With the method established in this paper, the work is done simply, rapidly, sensitively and accurately. It was actually adapted to determine ERFX and it's metabolite CPFX concentrations in plasma and pharmacokinetic studies, and to determine ERFX and CPFX contents in calves plasma successfully for the first time.