The overall goals of the broad and growing field of molecular medicine is to identify fundamental errors of disease and to develop corrections of them on the molecular level. At the same time, real-time imaging of gene expression in vivo aims towards a detailed analysis of both endogenous and exogenous gene expression in animal models of disease and in the clinical setting. Non-invasive imaging of endogenous gene expression may reveal insight into the molecular basis of disease pathogenesis and the extent of treatment response. When exogenous genes are introduced, e.g. by herpes simplex virus type 1 (HSV-1)-based vectors, to ameliorate a genetic defect or to add an additional gene function to cells, imaging techniques may reveal the assessment of the location, magnitude and duration of therapeutic gene expression and its correlation to the therapeutic effect. Here, we review the main approaches of non-invasive imaging techniques of gene expression in vivo with special reference to HSV-1 vector-mediated gene expression.