The simple, rapid and sensitive liquid chromatographic separation of five macrolides (tilmicosin, erythromycin, tylosin, roxithromycin and josamycin) widely used in food producing animals was developed. Response surface methodology was used as an optimization method of mobile phase, column temperature and pH to provide the best resolution of these analytes. The separation was performed by using an end-capped X-Terra RP-18 column (250 x 4.6 mm I.D x 5 m) with an isocratic system of 15 mM hydrochloric acid (pH 2.5)-acetonitrile as the mobile phase at a temperature of 30A degrees C and flow-rate of 1.0 mL/min. The suitability of the method for multi-residue determination of the macrolides is demonstrated by the analysis of milk samples spiked with tylosin. Roxithromycin was used as internal standard. The recovery of tylosin was quite good as 90.8%. The limit of quantification and detection limit were 0.024 and 0.007 mu g/mL, respectively. The method was successfully applied to determination of macrolides at levels below the maximum concentration legally allowed in milk samples.