Over 10 years ago, the first successful gene therapy paradigms for experimental brain tumors models have been conducted, and they were thought to revolutionize the treatment of patients with gliomas. Application of gene therapy has been quickly forced into clinical trials, the first patients being enrolled in 1994, with overall results being disappointing. However, single patients seemed to benefit from gene therapy showing long-term treatment response, and most of these patients bearing small glioblastomas. Whereas the gene therapy itself has been performed with high sophistication, limited attention has been paid on technologies, which (i) allow an identification of viable target tissue in heterogenous glioma tissue and which (ii) enable an assessment of successful vector administration and vector-mediated gene expression in vivo. However, these measures are a prerequisite for the development of successful gene therapy in the clinical application. As biological treatment strategies such as gene and cell-based therapies hold promise to selectively correct disease pathogenesis, successful clinical implementation of these treatment strategies rely on the establishment of molecular imaging technology allowing the non-invasive assessment of endogenous and exogenous gene expression in vivo. Imaging endogenous gene expression will allow the characterization and identification of target tissue for gene therapy. Imaging exogenously introduced cells and genes will allow the determination of the 'tissue dose' of transduced cell function and vector-mediated gene expression, which in turn can be correlated to the induced therapeutic effect. Only these combined strategies of non-invasive imaging of gene expression in vivo will enable the establishment of safe and efficient vector administration and gene therapy protocols for clinical application. Here, we review some aspects of imaging in gene therapy trials for glioblastoma, and we present a 'proof-of-principle' 2nd-generation gene therapy protocol integrating molecular imaging technology for the establishment of efficient gene therapy in clinical application.