Transposons present an effective alternative to viral gene delivery that avoids the adverse events of viral vectors (e.g., neutralizing antibodies, immune responses, and insertional mutagenesis). During the last decade, several transposon systems have been developed to deliver therapeutic genes or to correct defective genes in mammalian cells to treat monogenetic and complex diseases such as hemophilia A and B, leukemia, or age-related macular degeneration. Numerous eukaryotic cells have been efficiently transfected including muscle, hematopoietic, ocular, neural, and stem cells. Since transposons integrate the gene of interest stably, but without preference to transcriptional active sites into the host cell’s genome, they offer life-long therapeutic options with much lower risk of insertional mutagenesis compared to integrative viral vectors. Transposons can be used to deliver genes in vivo locally, systemically, and ex vivo to cells for transplantation. Recently, the safety of transposon-based gene delivery has been enhanced by the use of transposases delivered as mRNA or protein, cell-specific promotors, free-of-antibiotic resistance genes and mini-plasmids, and targeted integration. GMP-grade plasmid production for human use has been established and several gene therapeutic treatments using transposons are already in clinical testing. This methods collection aims to present state-of-the-art techniques for the production of transposon plasmids, delivery techniques, and applications. Scientists from multidisciplinary fields of research will share their knowledge of the potential of transposon-based gene delivery for the treatment of several diseases, as well as strategies to improve transposon-based gene delivery