To develop a successful protein therapeutic, effective DNA delivery technologies are required that induce high and sustained levels of protein production in appropriate targets sites, whereas robust and long-lasting immune responses need to be induced by a DNA-based vaccine. Vectors for gene therapy and DNA vaccines must be resistant to degradation and attack by the immune system, have a satisfactory safety profile, and be able to express the therapeutic protein for the desired period of time. Effective non-viral vectors, which can express the proteins of interest at high levels, are available. However, since most of the DNA delivered in vivo is degraded before it can enter the nucleus, proper formulation and delivery are critical to the development of effective gene-based therapeutics and vaccines. These systems must be safe for human and veterinary clinical applications and yet ensure that the DNA survives the extra- and intracellular environment and is capable of entering the appropriate cellular compartments. In this review various potential and proven non-invasive chemical, mechanical, physical and biological DNA delivery systems for therapeutic and vaccine applications are discussed. A few of these approaches have been evaluated and proven to be promising in target species. Others, which promise to be less invasive, have only just started to be explored.