A simple sample preparation technique coupled with the specific and sensitive fluorescence detection for the determination of moxifloxacin in human plasma was developed and fully validated. Levofloxacin was chosen as an internal standard. Chromatographic separation was achieved using a Shiseido C18 MGII (250×4.6mm i.d.; 5 μm) column under an isocratic mobile phase comprising of 50 mM potassium dihydrogen phosphate (pH 2.4) - acetonitrile (77:23, v/v) at a flow rate of 1.5 mL/min. Fluorescence detection was optimized for the determination of moxifloxacin in human plasma at an excitation wavelength of 296 nm and an emission wavelength of 504 nm. The total chromatographic run time was 8 min with the retention times of moxifloxacin and internal standard at 6.5 and 3.0 min, respectively. Calibration curves were established over the dynamic range of 20-3000 ng/mL. The analytical method was validated as per US-FDA and EMA guidelines for specificity, sensitivity, linearity, accuracy, precision, recovery, hemolytic effect, lipemic effect, dilution integrity and stability. The validated analytical method was successfully applied in a pharmacokinetic study of a single-dose oral administration of a moxifloxacin 400 mg tablet in Thai healthy volunteers.