Gene-directed enzyme prodrug therapy (GDEPT) is one of the promising alternatives to conventional chemotherapy. Suicide gene therapy based anticancer strategy involves selective introduction of a foreign gene into tumor cells to produce a foreign enzyme that can activate an inert prodrug to its cytotoxic form and cause tumor cell death. In this review, we present three most promising suicide gene/prodrug combinations (1) herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV), (2) cytosine deaminase (CD) from bacteria or yeast with 5-fluorocytodine (5-FC) and (3) bacterial nitroreductase (NTR) with 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954) and discuss how molecular imaging may improve therapy strategies. Current advances in noninvasive imaging technologies can measure vector dose, tumor selectivity, transgene expression and biodistribution of therapeutic gene with the aid of reporter genes and imageable probes from live animal. In this review we will discuss various imaging modalities - Optical, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), and highlight some of the approaches that can advance prodrug cancer therapy from bench to clinic.