The traditional approach for determination of differences in unbound drug concentrations between normal and special human populations, e.g., those with renal or hepatic impairment, follows an ex vivo study design. This approach entails the collection of blood samples from human subjects following administration of a candidate drug and subsequent analysis of isolated plasma for unbound drug concentration. However, there are several shortcomings with this approach. While much attention is given to sample analysis using a validated analytical method, clinical samples are often prepared using unsuitable procedures. In addition, the inability to predict or control the concentration of drug in post-dose plasma samples often results in unbound fraction below the limit of quantitation. Furthermore, the traditional ex vivo approach does not adequately factor drug concentration dependence on plasma protein binding. Therefore, the use of typical ex vivo data in pharmacokinetic analyses may be questionable when unbound fractions vary with total plasma concentrations. Here, we present a simple in vitro approach to determine unbound drug concentrations involving special populations based on the accepted practice used in normal human populations. This accepted approach begins with fortifying plasma with a known range of drug concentrations to establish relationship between drug concentration and unbound drug in plasma. This concentration-dependence relationship is applicable to subsequent clinical studies and leads to more informative data for pharmacokinetic analyses. This same strategy applies to special human populations, where pre-dose plasma from each subject is fortified with known concentrations of the drug, thereby offering several advantages. For example, precise control of in vitro plasma drug concentrations ensures that the measured unbound drug concentration will fit the quantifiable range of the analytical method. In addition, this approach can use a radiolabeled form of the drug, even when a nonradiolabeled form is used in the clinical study. The use of a radiolabeled form of the drug improves analytical accuracy and provides a more definitive measure of non-specific binding. We present in vitro data demonstrating good correlation with ex vivo data for accurate characterization of unbound drug concentrations, thereby verifying the in vitro approach for use with normal and special populations.