Recent regulatory guidance suggests that drug metabolites identified in human plasma should be present at equal or greater levels in at least one of the animal species used in safety assessments (MIST). Often synthetic standards for the metabolites do not exist, thus this has introduced multiple challenges regarding the quantitative comparison of metabolites between human and animals. Various bioanalytical approaches are described to evaluate the exposure of metabolites in animal vs. human. A simple LC/MS/MS peak area ratio comparison approach is the most facile and applicable approach to make a first assessment of whether metabolite exposures in animals exceed that in humans. In most cases, this measurement is sufficient to demonstrate that an animal toxicology study of the parent drug has covered the safety of the human metabolites. Methods whereby quantitation of metabolites can be done in the absence of chemically synthesized authentic standards are also described. Only in rare cases, where an actual exposure measurement of a metabolite is needed, will a validated or qualified method requiring a synthetic standard be needed. The rigor of the bioanalysis is increased accordingly based on the results of animal:human ratio measurements. This data driven bioanalysis strategy to address MIST issues within standard drug development processes is described.