An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 mum (25 cm x 4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30 degreesC and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile - 0.25 M tetrabutylommonium hydrogen sulfate pH 75 - 0.25 M ethylenediaminetetraacetic acid pH 7.5 - water (115:360:160:365, v/v/v/v) is used at a flow rate of 1.0 mL.min(-1), to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity repeatability linearity and sensitivity.