Over the last three decades, imaging has been a thriving field with continuous egression of more reliable and highly sophisticated tools and techniques allowing better understanding of biological processes in living organisms. This field continues to expand and its applications broaden to encompass limitless applications in various biomedical research areas. It is however, of utmost importance to understand the capabilities and limitations of this technique as new challenges and hurdles continue to arise. This chapter describes the general properties of bioluminescence imaging and commonly used reporters while underlining the challenges and limitations with these modalities.