A coupled column liquid chromatography system was applied for the separation of the burnup monitors in spent nuclear fuel sample solutions. A reversed phase column was studied for the adsorption behavior of uranyl ions using alpha-hydroxyisobutyric acid as an eluent and used for the separation of plutonium and uranium. A cation exchange column prepared by coating 1-eicosylsulfate onto the reversed phase column was used for the separation of the lanthanides. In addition, retention of Np was checked with the reversed phase column and cation exchange column, respectively, according to the oxidation states to observe the interference effect for the separation of burnup monitors. This chromatography system showed a great reduction in separation time compared to a conventional anion exchange method. A good agreement from the burnup data was obtained between for this method and a conventional anion exchange method to within 1% of a difference for the spent nuclear fuel samples of about 40 GWD/MTU.