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161 Tb is a promising radionuclide, which is a suitable radionuclide with favorable properties for small treatment size of cancer. NCA 161 Tb can be produced by the indirect method through 160 Gd(n,γ) 161 Gd→ 161 Tb nuclear reaction. To obtain the NCA radionuclides, the existence of an effective Gd/Tb separation method is critical. In this study, isolation of Tb from Gd/Tb matrix using Ln resin column based on extraction chromatography method has been carried out. Fractions eluted from the column containing Gd/Tb matrix were identified and quantified using ICP. The optimization of different experimental parameters for the effective separation of Gd/Tb, such as concentration of eluting solutions and flow rate of load and elution, was investigated. The results showed that optimum Gd/Tb isolation condition was obtained using HNO3 solution with a concentration of 0.8 and 3 N to separate gadolinium and terbium isotopes, respectively. The separation yield of Tb and Gd was obtained at 83.51 % and 81.8%, respectively.