Recent progress in the development of non-invasive imaging technologies continues to strengthen the role of molecular imaging in biological research. Small animal imaging with bioluminescence has been validated recently in a variety of research models and have been shown to provide continuous quantitative monitoring of the location(s), magnitude, and time-variation of gene delivery and/or expression. This article reviews the role of In Vivo bioluminescence imaging technologies as they have been used in imaging gene delivery and gene expression for molecular imaging applications. The studies published to date demonstrate that bioluminescence imaging tools will help to understand human diseases through small animal models.