This paper presents a simple, rapid, and sensitive method of investigating the pharmacokinetics of zabofloxacin, a fluoroquinolone antibiotic agent used to treat infections caused by multidrug-resistant Gram-positive pathogens. The method presented involves the use of high-performance liquid chromatography with ultraviolet (UV) detection without an internal standard. Zabofloxacin was separated using an isocratic elution on a Capcell Pak C18 column using an acetonitrile–methanol–phosphate buffer (1 g of KH2PO4and 1 g of heptane sulfonic acid sodium salt in 720 mL of purified water) and a 1 M tetrabutylammonium dihydrogenphosphate solution (18.5:8.5:72:1, by volume) as a mobile phase at a flow rate of 0.25 mL/min with UV detection at 275 nm. The lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) were 100 ng/mL and 20000 ng/mL, respectively, with acceptable linearity in the range from 100 to 20000 ng/mL (R > 0.999). The intra- and inter-day accuracy (RE) ranged from −8.2% to 1.8% and the intra- and inter-day precision (CV) ranged from 3.8% to 10.6% for zabofloxacin. In addition, stock solution stability, recovery, freeze–thaw effects, and short-term and long-term stability met the acceptance criteria. Following oral administration of zabofloxacin to rats at a dose of 250 mg/kg, the concentration of zabofloxacin was readily quantifiable in plasma samples up to 8 hr post-dose. This suggests that a validated assay can be used to study the pharmacokinetics of zabofloxacin in rats. [ABSTRACT FROM PUBLISHER]