Bioluminescent imaging (BLI) is a powerful noninvasive tool that has dramatically accelerated the in vivo interrogation of cancer systems and longitudinal analysis of mouse models of cancer over the past decade. Various luciferase enzymes have been genetically engineered into mouse models (GEMM) of cancer, which permit investigation of cellular and molecular events associated with oncogenic transcription, posttranslational processing, protein-protein interactions, transformation, and oncogene addiction in live cells and animals. Luciferase-coupled GEMMs ultimately serve as a noninvasive, repetitive, longitudinal, and physiologic means by which cancer systems and therapeutic responses can be investigated accurately within the autochthonous context of a living animal. Significance: Luciferase-dependent bioluminescence imaging coupled with genetically engineered mouse models of cancer permit interrogation of tumor biology and therapeutic response within the proper physiological context of the whole animal in vivo. © 2013 American Association for Cancer Research.