The invention provides compounds, compositions, methods, 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. luciferins or coelenterazines, that are cytochrome P450 substrates and that are also bioluminescent enzyme, e.g., luciferase, pro-substrates are provided. 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.

The present disclosure relates to methods for evaluating the interaction of a candidate compound on 3D hepatocyte spheroid in an invitro culture, including evaluating the metabolism of a candidate compound, for use in various biochemical and molecular biology studies. The methods are performed in labware that combine 3D spheroid culture with micro-patterned design that allows for multiple to several hundreds of spheroids to be treated under the same conditions and to produce sufficient materials (e.g., parent drug, drug metabolites, DNA, RNA, and proteins from cells) and higher detection signal intensity for ADME/Tox (absorption, distribution, metabolism, excretion and toxicity) studies. The methods allow for, among other uses, the investigation and generation of accurate invitro intrinsic clearance data and thus more accurate prediction of in vivo clearance, particularly with low clearance compounds.