Integral to the development of all gene therapy technologies is the ability to monitor gene delivery, in terms of distribution, levels and kinetics of vector transgene expression. This can be achieved to some extent at the preclinical level through use of traditional ex vivo analytical methods, but these hold several drawbacks, not least the requirement for death of experimental subjects for such end-point assays. Real-time in vivo analysis of reporter gene expression empowers the investigator with the ability to non-invasively assess gene delivery over time, as well as host responses to vector administration and therapeutic interventions. While there exist several technologies for such small animal monitoring, imaging of light emission from luminescent or fluorescent reporters has become the mainstay of preclinical imaging for gene therapy research. Optical imaging strategies represent powerful yet cost-efficient and convenient systems compared with alternative methods. Through tagging of vector and/or cells or interest with suitable reporter genes, both vector and host responses can be assessed in rapid, high-throughput analyses, providing spatial, temporal and quantitative read-out, without the need for radioactivity. In this review, we discuss the current state-of-the-art for optical technologies, describe related approaches employed in gene therapy research for a wide range of diseases, and outline the potential for this imaging modality in the progression of gene therapy as a medicine.