In this study, the combined effect of the organic modifier content and mobile phase pH has been used to predict the chromatographic behavior of methicillin, oxacillin, nafcillin, cloxacilin and dicloxacillin. The optimum separation condition of these drugs in acetonitrile-water binary mixture was determined from the mobile phase pH dependence of their retention factor. A Synergy Fusion-RP C-18 analytical column (150 mm x 4.6 mm I.D., 4 mu m) was preferred to carry out the developed method. Chromatographic measurements were done at 30 degrees C with an eluent flow rate of 1 mL min(-1). In order to validate the optimized conditions, these drugs were applied successfully in spiked samples of human urine. Valsartan was chosen as an internal standard. The described method was linear over a concentration range of 2-32 mu g mL(-1) and 3-48 mu g mL(-1) for the assay of methicillin, nafcillin, dicloxacillin and oxacillin, cloxacillin respectively. Good linear relationships over the investigated concentration ranges were observed with values of correlation coefficient higher than 0.999 for all of the drugs. The intra-day and inter-day precisions of this method were evaluated with RSD (%) values less than 0,561 and 1,622%, respectively. Optimized assay method was validated according to United States Pharmacopeia (USP) guidelines to confirm linearity, accuracy and precision.