As science progresses in its understanding of diseases and their treatment, advances have been made in the biotechnology used in disease therapy. Most gene therapy approaches utilise viral vectors to deliver genes of interest. However, multiple proteins are often involved in disease processes and there is often a need to efficiently deliver more than one gene. Researchers have employed several strategies to accomplish this goal. When designing vectors to express multiple genes, there are several factors that need to be taken into account, including cell type, the activity of the protein of interest and subcellular protein localisation. In most cases, it is ideal for each protein to be expressed at comparable levels, a leading issue with traditional strategies for multigene expression. This review describes some of the techniques that have been used to express multiple genes, and will focus on the use of 2A peptides or 2A peptide-like sequences in the design of multicistronic vectors that may alleviate some of these issues.