Determining the biliary clearance of drugs in humans is very challenging because bile is not readily accessible due to the anatomy of the hepatobiliary tract. The collection of bile usually is limited to postsurgical patients with underlying hepatobiliary disease. In healthy subjects, feces typically are used as a surrogate to quantify the amount of drug excreted via nonurinary pathways. Nevertheless, it is very important to characterize hepatobiliary elimination because this is a potential site of drug interactions that might result in significant alterations in systemic or hepatic exposure. In addition to the determination of in vivo biliary clearance values of drugs, the availability of in vitro models that can predict the extent of biliary excretion of drugs in humans may be a powerful tool in the preclinical stages of drug development. In this review, recent advances in the most commonly used in vivo methods to estimate biliary excretion of drugs in humans are outlined. Additionally, in vitro models that can be employed to investigate the molecular processes involved in biliary excretion are discussed to present an updated picture of the new tools and techniques that are available to study the complex processes involved in hepatic drug transport.