Cefpodoxime proxetil (CFP), a broad-spectrum third-generation cephalosporin, has been used most widely in the treatment of respiratory and urinary tract infections. For bioequivalence study of CFP in rabbit plasma, it was necessary to develop a highly sensitive and selective high-performance liquid chromatographic (HPLC) method with fluorescence (FL) detection. The pre-column labeling of cefpodoxime acid (CFA) (active metabolite) with an efficient benzofurazan type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was carried out in the present study in 100 mM borate buffer (pH = 8.5) at 50°C for 15 min. The obtained fluorescent products were separated on C18 column with an isocratic elution of the mobile phase, which consists of 10 mM phosphate buffer(pH = 3.5)/CH3CN (70:30, v/v). The fluorescent product (DBD-CFA) was detected fluorimetrically at 556 nm with an excitation wavelength of 430 nm. Cefotaxime sodium was used as internal standard. The method was validated according to the requirements of US-FDA guidelines. The correlation coefficient of 0.999was obtained in the concentration ranges of 10-1000 ng mL-1. The limits of detection and quantification (S/N = 3) were 3 and 10 ng mL-1, respectively. Plasma CFA levels were successfully determined in rabbit with satisfactory precision and accuracy. The proposed HPLC-FL method was successfully applied to study bioequivalence in rabbits for two formulations of different brands contained CFP (prodrug) in a randomized, two-way, single-dose, crossover study and all pharmacokinetic parameters for the two formulations were assessed.