The analysis of circulating metabolites is driven by the need to complete a comprehensive PK/PD exploration of the activity of a drug. When detected and identified, circulating metabolites should be considered from their concentration, structure (relationship to parent and known structure-activity relationships) and physicochemistry (in particular lipophilicity, polar surface area and charge). All these considerations help to determine the likelihood of the metabolites exerting on and off target pharmacology, which may contribute to the overall efficacy and side effects (including toxicity) of the drug. Circulating metabolites do not directly allow an estimate of the actual amounts formed (or proportion of the dose) of a particular metabolite. The amount formed is useful to estimate the chances of possible toxicity, in clinical use, from reactive metabolites. Such metabolites are not usually detectable in their reactive, unstable form. They are readily characterized, however, and quantified as downstream excreted metabolites such as glutathione conjugates. Clinical use of a drug is often specified for special populations (polymorphic drug metabolizing enzymes) and co-medications (drug-drug interactions). The early investigation of these uses in vitro enzymology assessments. It is important to ensure that the in vitro pathways match those seen in humans in vivo. Assignment of clearance routes is facilitated by assessment of the proportion of parent drug cleared by the various routes using excreted metabolite data.