Molecular imaging techniques have become an essential tool in drug discovery because they provide critical insights into drug penetration and efficacy using non-invasive approaches. Several different molecular imaging methods exist, including ultrasound, radionuclide imaging, magnetic resonance, and optical imaging. Optical imaging has grown in popularity because it is based on bioluminescence or fluorescence imaging techniques. Bioluminescence imaging (BLI) is well suited to drug discovery in animal models and in vitro systems because it does not require any radioactive reagents, has high molecular sensitivity with low background, and reagents are widely available and not especially costly. Here we highlight key features of BLI that makes this method a drug discovery essential.