A separation to the baseline isocratic technique has been developed for the evaluation of the purity of aprotinin by internal normalization. The column used was a 30 nm pore diameter butyl-bonded silica stationary phase. An exact relationship was found to give an adequate description of the retention of aprotinin using acetonitrile concentrations of 17-22% and NaClO4 concentrations of 5-50 mmol. It can be used to optimize the mobile phase content for the particular user column and to compensate for possible variations in the retention time of aprotinin, analysed on different batches of butyl-bonded stationary phases.