A single-stage dual-column chromatographic technique has been developed in this study for separation and determination of lanthanides in a uranium matrix. A 5-cm-length reversed-phase column coated with tri-n-octylphosphine oxide (TOPO) was connected in series to a 10-cm-length reversed-phase monolithic column (dynamically modified into a cation exchange column) to accomplish individual isolation of lanthanides from the uranium matrix. The proposed technique eliminates the step of uranium matrix removal for the determination of lanthanides. Samples with a lanthanide-to-uranium ratio (1 part lanthanide to 105 parts uranium) were directly injected into the dual column for the quantitative determination of lanthanides without uranium matrix removal. In some studies, samples of lanthanides in the uranium matrix could be injected as much as 45 times consecutively into a high-performance liquid chromatography system for determination of lanthanides without any uranium elution. The retention behavior of Pu(IV), Pu(III), Am(III), and fission products was also investigated on the TOPO-coated support. The single-stage dual-column chromatographic technique was demonstrated for the determination of fission products such as La and Nd in the dissolver solution of pressurized heavy water reactor spent fuel for the measurement of atom percent fission burnup. The technique can also be employed to estimate lanthanide impurities in samples of UO2 (1 part lanthanide to 106 parts uranium) without removal of the uranium matrix.