One of the major drawbacks in the analysis of aminoglycoside antibiotics is their lack of UV chromophore and/or fluorophore. Tobramycin, a representative member of this group, was examined in this study. To overcome the detection hurdle, a precapillary derivatization followed by capillary electrophoresis analysis with direct UV detection was investigated. A central composite design was applied to optimize the method and three parameters were selected in this study: buffer pH, temperature and % acetonitrile (ACN). Selectivity between tobramycin main component and its adjacent peaks as well as the peak efficiency and symmetry factors were established as responses. For each response, a model was obtained by a second-order mathematical expression. Successful results were obtained with a simple background electrolyte (BGE) containing 30 mM sodium tetraborate, pH 10.2, and ACN (75:25 v/v). Under these conditions, baseline separation of tobramycin from its adjacent kanamycin B and an unknown peak was achieved. A temperature of 20 degrees C and applied voltage of 28.0 kV were used. The method showed good validation data in terms of precision, limits of quantitation and detection, specificity and linearity and was found to be suitable for analysis of tobramycin bulk pharmaceutical samples.