The present invention provides methods, compositions, substrates, and kits useful for analyzing the 
metabolic activity in cells, tissue, and animals and for 
screening test compounds for their effect on 
cytochrome P450 activity. In particular, a one-step and two-step methods using luminogenic molecules, e.g. 
luciferin or coelenterazines, that are 
cytochrome P450 substrates and that are also bioluminescent 
enzyme, e.g., 
luciferase, pro-substrates are provided. Upon addition of the 
luciferin derivative or other luminogenic molecule into a P450 reaction, the P450 
enzyme metabolizes the molecule into a bioluminescent 
enzyme substrate, e.g., 
luciferin and / or luciferin derivative 
metabolite, in a P450 reaction. The resulting 
metabolite(s) serves as a substrate of the bioluminescent enzyme, e.g., 
luciferase, in a second light-generating reaction. Luminescent 
cytochrome P450 assays with low background signals and high sensitivity are disclosed and isoform selectivity is demonstrated. The present invention also provides an 
improved method for performing 
luciferase reactions which employs added 
pyrophosphatase to remove inorganic 
pyrophosphate, a luciferase inhibitor which may be present in the reaction mixture as a contaminant or may be generated during the reaction. The 
present method further provides a method for stabilizing and prolonging the luminescent 
signal in a luciferase-based 
assay using luciferase 
stabilizing agents such as reversible luciferase inhibitors.