A reversed-phase higk-performance liquid chromatographic method is described for the determination of seven quinolones in plasma and amniotic fluid. Experimental designs have been applied for the optimization of the method in order to determine the experimental conditions for maximized resolution and minimized retention time. A total desirability function D that weights the responses together was used to optimize the two different responses simultaneously. The experimental responses were fitted into a second order polynomia. The optimum assay conditions were: 15 mM citrate buffer, pH 3.2, 9% MeCN, 5% MeOH, 5 mM TMAB, 1.5 ml. min(-1) flow rate and 40 degrees C column temperature. A simple and efficient solid phase extraction has been applied for preparation of samples. The recovery of quinolones is >95%. The optimized assay condition was validated according to Federal Drug Administration (FDA) guidelines to confirm specificity, linearity, accuracy, precision and robustness. The method developed has been applied to quantification of levofloxacin and moxifloxacin in amniotic fluid and plasma.