The study of enzyme kinetics in drug metabolism involves assessment of rates of metabolism and inhibitory potencies over a suitable concentration range. In all but the very simplest in vitro system, these drug concentrations can be influenced by a variety of nonspecific binding reservoirs that can reduce the available concentration to the enzyme system under investigation. As a consequence, the apparent kinetic parameters that are derived, such as K m or K i, can deviate from the true values. There are a number of sources of these nonspecific binding depots or barriers, including membrane permeation and partitioning, plasma or serum protein binding, and incubational binding. In the latter case, this includes binding to the assay apparatus, as well as biological depots, depending on the characteristics of the in vitro matrix being used. Given the wide array of subcellular, cellular, and recombinant enzyme systems utilized in drug metabolism, each of these has different components that can influence the free drug concentration. The physicochemical properties of the test compound are also paramount in determining the influential factors in any deviation between true and apparent kinetic behavior. This chapter describes the underlying mechanisms determining the free drug concentration in vitro and how these factors can be accounted for in drug metabolism studies, illustrated with case studies from the literature.