Improvements in gene delivery methods have primarily addressed the limitations of gene therapy, thereby enabling its application in areas such as oncology, vaccine development, regenerative medicine, and the correction of genetic disorders. In the past, viral vectors have been the primary choice in clinical trials due to their high efficiency of gene transfer; however, issues related to immunogenicity, genotoxicity, and difficulty in production have led to the explorations of non-viral vector options. Non-viral means of gene delivery, such as lipids, polymers, inorganic nanoparticles, and hybrid systems, offer a more secure and easily modifiable tool for gene delivery, with desirable properties including payload flexibility, scalability, and biocompatibility. Major innovations, such as lipid nanoparticle (LNP)-based mRNA vaccines for COVID-19 and CRISPR/Cas9 delivery through polymers, have opened new possibilities for their use in clinical practice. Nevertheless, several issues remain, including transfection efficiency, targeted delivery, and sustained expression. This chapter provides a detailed survey of the design, operations, and applications of non-viral gene delivery systems, with an emphasis on clinical scenarios and current and future technologies. In addition, it identifies the primary obstacles and proposes next steps to enhance the translational feasibility of non-viral vectors in precision medicine.