The better and earlier a disease can be diagnosed and characterized, the higher the chance of being able to interfere in this process with a chemical entity. This is the rationale for the use of in vivo molecular imaging techniques in pharmaceutical research and development. The present review addresses the value of fluorescence optical imaging in this area. Through the administration of exogenous fluorochromes, molecular processes can be monitored non-invasively in vivo with operational simplicity, safety and high cost-effectiveness. This provides potential for obtaining information related to target validation, detection and characterization of pathology, and treatment evaluation. Although most applications are preclinical, in small rodents, a few clinical uses are also emerging. A key element for the success of the technique is the availability of an adequate fluorescent probe to address a given biological or pharmacological question of interest.