The separation of enzymatic hydrolysis products of yeast RNA by ion-pair HPLC was studied. A modified chromatographic response function (MCRF) was proposed to appraise the effectiveness of chromatographic separation. This function takes the number of peaks, resolution and the retention time of the last peak into consideration. It shows advantages for optimization of HPLC separation of complex mixtures. An orthogonal array design was used to separate the hydrolysate of yeast RNA and the optimal chromatographic conditions were obtained.