Assessing the interactions of a new drug candidate with transporters, either as a substrate or as an inhibitor, is no simple matter. There are many clinically relevant transporters, as many as nine to be evaluated for an FDA submission and up to eleven for the EMA as of 2013. Additionally, it is likely that if a compound is a substrate or inhibitor of one transporter, it will be so for other transporters as well. There are practically no specific substrates or inhibitors, presumably because the specificities of drug transporters are so broad and overlapping, and even fewer clinically relevant probes that can be used to evaluate transporter function in humans. In the case of some transporters, it is advisable to evaluate an NCE with more than one test system and/or more than one probe substrate in order to convince oneself (and regulatory authorities) that a clinical drug interaction study is not warranted. Finally, each test system has its own unique set of advantages and disadvantages. One has to really appreciate the nuances of the available tools (test systems, probe substrates, etc.) to select the best tools for the job and design the optimal in vitro experiment. In this chapter, several examples are used to illustrate the successful interpretation of in vitro data for both efflux and uptake transporters. Some data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in transporter kinetics to the reader.