While the application of radionuclide generators in nuclear medicine constitutes a successful example of the availing of short lived radionuclides on demand, the success and growth of generator technology largely depend on the progress in separation science. Separation science plays a pivotal role in the development of radionuclide generators, with this branch of science being in a process of continuous evolution. Any breakthrough, technology innovation, or improvement over existing paradigms in separation science represent not only an important driving force but also lay the cornerstone for further accomplishments in radionuclide generator technology. With the emergence of professionally run central radiopharmacies, radionuclide generator technology is poised to undergo a game change in terms of design and user profiles. It is of utmost importance to assure the access to appropriate radionuclide generator systems that are adaptable to the existing and foreseeable demands of the nuclear medicine community. The purpose of this review is to assess the recent advances in separation science which can act as a springboard to spur further development in radionuclide generator technology. Such advances are expected to unfold a wide spectrum of radionuclide generators to accelerate further development in this field. In light of this, a variety of separation technologies that have traditionally been used and emerging techniques that have the potential to promote rapid growth in generator technology have been selected, highlighted, and critically evaluated to illustrate their impact on the development of clinically useful radionuclide generators. The advantages, limitations, and potential applications of the separation methods, along with their current status and future prospective in radionuclide generator development, are discussed.