Human and mouse cancer cell lines are used in xenograft and syngeneic models, respectively, for studying in vivo tumor formation and development, evaluating metastasis, measuring tumor burden in whole animals, and monitoring response to therapeutic treatment. Whole animal in vivo imaging has been widely applied by researchers due to the ease of operation in visualizing in vivo biological events while eliminating the requirement for animal subject sacrifice, allowing for continual monitoring/imaging of a single individual animal, and reducing the amount of inter-animal variation. Luciferase reporters provide a relatively simple, robust, and highly sensitive means to measure biological processes and to assess drug efficacy in animal models through in vivo bioluminescence imaging. Here, we report on the generation of a panel of cell lines which express high levels of luciferase and have broad applications for in vitro and in vivo studies. The panel includes human and mouse cell lines that represent various common cancer and tissue types such as breast, colon, lung, pancreas, prostate, and skin. After introduction of a Lenti-LUC2 luciferase reporter into the parental cell lines, single cell cloning was performed to isolate stable clones with high luciferase expression. We quantified in vitro bioluminescence signals within the cells and analyzed the signal-to-noise ratio. In addition, the cell lines were characterized and authenticated by using cell morphology, growth kinetics, and STR analysis. Luciferase-labeled human cell lines were administered into immune-deficient mice either by subcutaneous or orthotropic injection to establish xenograft models. Luciferase labeled mouse cell lines were tested in the associated breed of mice to evaluate tumor growth in the syngeneic mouse model. The bioluminescence live imaging was performed using the Xenogen IVIS® imaging system and the in vivo bioluminescent radiance was analyzed. Although growth can be evaluated by both traditional caliper measurement and bioluminescence in subcutaneous mouse models, only live bioluminescent imaging enables continuous monitoring tumor metastasis or analysis of tumor growth of the intracranial brain tumor models. In summary, luciferase-expressing cell lines are valuable tools for elucidating mechanisms involved in tumorigenesis, studying tumors in vivo, and screening anti-cancer compounds for drug discovery and development.