Directed evolution (DE) is a process of mutation and artificial selection to breed biomolecules with new or improved activity. DE platforms are traditionally prokaryotic or yeast-based, and stable mutagenic mammalian systems have been challenging to establish and apply. Issues with recombination and safety for virus-derived approaches are a concern as well as maintenance of the system in host cells. To this end, we developed PROTein Evolution Using Selection (PROTEUS), a platform that uses chimeric virus-like vesicles to enable extended mammalian DE campaigns without system collapse. This platform can generate sufficient diversity for DE in mammalian systems. Using PROTEUS, we engineered the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation. PROTEUS is also compatible with intracellular nanobody evolution, and we apply it to design a DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is an efficient and lasting platform to directionally evolve biomolecules of interest within mammalian cells, with applications in biotechnology, therapeutics development and beyond.