The development of novel drugs is a lengthy process requiring years of preclinical research and many steps indispensable to ensure that the molecule of interest can be administered to humans with a minimal risk of toxic effects. Even a minimal reduction in the initial stages of drug development would result in a tremendous saving in time; therefore, pharmaceutical companies are eager to apply novel methodologies that shorten the time required for pharmacodynamic, pharmacokinetic and toxicological studies to be carried out in vitro and in animal systems. Currently, quantitative analysis of molecular events in living organisms is done with the combined application of imaging and genetic engineering technologies. In vivo imaging provides surrogate endpoints that can improve the identification of new drug candidates and speed up their research at preclinical stages. The integration of reporter systems in animal models of human diseases represents a reachable frontier that will dramatically advance drug development in terms of costs, time and efficacy. The present review outlines the applicability of imaging technologies for drug development and presents a panorama on the state of the art of currently available imaging technologies suitable for preclinical studies, with particular focus on bioluminescence and fluorescence as the methodologies of election.