The estimation of Pu/²⁴¹Am ratio in the biological samples is an important input for the assessment of internal dose received by the workers. The radiochemical separation of Pu isotopes and ²⁴¹Am in a sample followed by alpha spectrometry is a widely used technique for the determination of Pu/²⁴¹Am ratio. However, this method is time consuming and many times quick estimation is required. In this work, Pu/²⁴¹Am ratio in the biological sample was estimated with HPGe detector based measurements using gamma/X-rays emitted by these radionuclides. These results were compared with those obtained from alpha spectroscopy of sample after radiochemical analysis and found to be in good agreement.