Gene therapy is a very attractive strategy in experimental cancer therapy. Ideally, the approach aims to deliver therapeutic genes selectively to cancer cells. However, progress in the improvement of gene therapy formulations has been hampered by difficulties in measuring transgene delivery and in quantifying transgene expression in vivo. In clinical trials, endpoints rely almost exclusively on the analysis of biopsies by molecular and histopathological methods, which provide limited information. Therefore, to ensure the rational development of gene therapy, a crucial issue is the utilisation of technologies for the non-invasive monitoring of spatial and temporal gene expression in vivo upon administration of a gene delivery vector. Such imaging technologies would allow the generation of quantitative information about gene expression and the assessment of cancer gene therapy efficacy. In the past decade, progress has been made in the field of in vivo molecular imaging. This review highlights the various methods currently being developed in preclinical models.