There has recently been an explosion in the availability of new technologies to noninvasively detect biological processes in preclinical models. One such modality, optical imaging, comprises using bioluminescent and fluorescent reporters and probes to repetitively interrogate molecular events and monitor disease progression in animal models. This review includes an overview of optical imaging technologies (e.g., hardware, reporters, probes) available for small animal imaging and their application in monitoring disease progression, therapeutic efficacy, and molecular processes such as proliferation, apoptosis, and angiogenesis. Also discussed are some of the challenges associated with in vivo optical imaging and the necessary controls and biological correlates one must include in experimental design and interpretation for successful preclinical studies.