A capillary zone electrophoresis method has been developed for the direct determination of norfloxacin in the physiological perfusate of isolated rat liver. Norfloxacin and the internal standard triamterene were detected using laser-induced fluorescence (LIF) detection with the excitation and emission wavelength of 325 and 435 nm, respectively. The background electrolyte (BGE) was 50 mM phosphate buffer (pH 4.6). The effect of pH and concentration of BGE on the electrophoretic migration and fluorescence response of analytes were examined. Calibration curves were linear over a wide range of 0.01–100 μg/mL. The limit of quantitation was 0.01 μg/mL. The intra- and inter-day relative standard deviation was 3.7%, or less, and the accuracy was 93.2% of the nominal concentration. No endogenous substances were found to interfere. The method was used to characterize the steady-state and transient pharmacokinetics of norfloxacin in the rat liver.