Zemuron is an amino steroid used as a muscle relaxant during anesthesia. Zemuron was analyzed by mixed-mode chromatography on a Primesep C HPLC column. Compound is hydrophobic and basic in nature, and retention time on cation-exchange mixed-mode column is controlled by the amount of acetonitrile, buffer concentration and buffer pH. In order to observe cation-exchange interaction on a Primesep C column, mobile phase pH should be maintained between 3-7.