The potential for various natural products to perturb the metabolism and disposition of medications has been recognized for decades. There are numerous in vitro and in vivo methods available to screen botanical products for drug interaction potential. Although many normal volunteer botanical-drug interaction studies have been performed, clearly, in vitro studies assessing the potential for drug interactions with various natural products represent the predominant type of published research performed to date. In addition to the recognized limitations of in vitro screening methodologies to assess conventional drug interactions, further difficulties emerge when examining botanical products. Primary challenges include assigning hepatic concentrations and accounting for bioavailability, distribution, first-pass metabolism and active metabolites. Additionally, variability in the chemical composition of commercially available botanical supplements, the lack of analytical standards and the inability to accurately screen the entities as mixtures add to complexities in experimental design. This mini-review is intended to address the particular problems and challenges in evaluating botanical supplements using in vitro methods, and review what can and cannot be learned from such investigations.