BioLuminescent OptoGenetics (BL-OG) tools, molecular reagents that utilize light emitted by a luciferase when interacting with its luciferin to activate light-sensing molecules, have been employed in animal models including C. elegans, zebrafish, mice, rats, and marmosets. This brief overview chapter highlights important parameters to be considered for in vivo applications of BL-OG. Routes of luciferin administration can be as simple as adding it to the food source for worms to applying it via several different routes in mammals, including intracranial, intravenous, and intraperitoneal injections. The choice of a particular route of administration in mammals will be affected by the luciferase–luciferin combination used and the kinetics desired. The choice of luciferin is determined by the luciferase employed in a BL-OG tool, with some luciferases offering a variety of luciferins optimized for in vivo applications and with varying signal stability. This overview chapter is followed by a chapter reviewing reports of BL-OG applications in therapeutic and basic science contexts in animal models. The second chapter highlights results from systematic studies of the relationships between bioluminescent light production, neural spiking activity, luciferin concentrations and injection parameters in the mammalian brain.