Ion-exchange chromatography (IEC) is the most frequently used chromatographic technique for the separation of proteins and peptides. In this article, the effects of urea on IEC separation of kiwifruit actinidin, egg white and urinary proteins were examined. The purity and relative amount of each protein in different conditions (in the presence or absence of urea) were compared with each other. The three parameters, including resolution, selectivity and efficiency of column in the presence of urea, were calculated and compared with the absence of urea. The results revealed that urea improved the purity of proteins and the resolution, selectivity and efficiency of IEC in separation of studied proteins.