Gene therapy or the delivery of genetic material can be carried out not only with viral vectors, but with non-viral vectors too. Although the use of non-viral carriers is safer, this approach has generally been poorer in terms of transfection efficiency. In order to improve the transfection rates, it is important to exactly know the path that the nanoparticles go through. In this way, we can understand the reason why some of these vectors fail or are successful in their duties. This review aims to summarize the different stages and barriers that nanocarriers have to overcome during endocytosis so as to reach the cytoplasm, in the case of RNA, or the nucleus, in the case of DNA. In addition, different strategies that could be employed to improve the success of the carriers in each stage are suggested.