A valuable strategy for the assessment of in vitro systems is proposed which involves a tiered approach consisting of four levels valuable for both selection of probe compounds and designing experiments for evaluation. At level 1, a Preliminary Assessment is used to triage novel systems based on existing information generated using the methods employed in the development of the system. There is no special requirement for specific probes or experimental conditions. At level 2, Metabolic and Transporter Competence and Cellular Integrity are investigated with a number of specific probes which are generally accepted as appropriate. The information obtained at this level (as with level 1) is largely qualitative in nature. At level 3, Quantitative Utility is established by kinetic studies conducted with specific probes under standard conditions of linearity with respect to time and protein concentration. It is essential that the latter be adhered to if subsequent scale up of the output metrics for uptake and clearance are to have appropriate (scalable) units. Finally level 4, Predictive Utility, is the most detailed level of evaluation involving several model compounds for which in vivo correlates are available. Model compounds have been collated that cover a wide range of metabolic clearance values, and it is important that comparisons are made with existing in vitro systems in order to show the added value of a novel approach including modelling and familiarity with in vivo investigations. © 2012 Elsevier Ltd. All rights reserved.