Concentrations of fluoroquinolones, which are used in the treatment of many bacterial infections, should be monitored in biological fluids as they exhibit concentration-dependent bactericidal activity. In this study, a liquid chromatography method for the determination of levofloxacin, ciprofloxacin, moxifloxacin and gemifloxacin in human urine and plasma was developed for the first time. The efficiency of five different columns for the separation of these fluoroquinolones was compared. Experimental parameters that affect the separation, such as percentage of organic solvent, pH, temperature, gradient shape and detector wavelength, were optimized by a step-by-step approach. Using a pentafluorophenyl core-shell column (100 x 4.6 mm, 2.7 mu m), the separation of four analytes was accomplished in <7.5 min. The developed method was validated for the determination of analytes in both urine and plasma with respect to sensitivity, specificity, linearity (r >= 0.9989), recovery (79.46-102.69%), accuracy, precision and stability (85.79-111.07%). The intra- and inter-day accuracies were within 89.55-111.94% with relative standard deviations of 0.35-8.05%. The feasibility of method was demonstrated by analyzing urine and plasma samples of patients orally receiving levofloxacin, ciprofloxacin or moxifloxacin. The developed method is suitable for therapeutic drug monitoring of these fluoroquinolones and can be applied to pharmacokinetic and toxicological studies.