Although plasma/serum is the preferred matrix for the characterization of pharmacokinetic parameters, recent years have witnessed the emergence of bile matrix as another tool for refining the pharmacokinetic disposition of drug(s) and the associated metabolite(s). The biliary excretion mechanism represents an important path for drug elimination through feces. Also, there are numerous examples in which bile samples have been shown to concentrate both drug and its metabolite(s) in a much higher proportion as compared with the circulating blood levels and may act as a reservoir for the re‐entry of the drug and its metabolite(s) to the systemic circulation once the bile gets drained into the small intestine.
Firstly, the review provides a comprehensive overview of various analytical methods that have been adopted for bile sample analysis with a description of extraction steps, chromatography and validation protocol. Secondly, it provides a discussion on bioanalytical related strategies including bile sample collection requirements. Thirdly, a brief discussion on fit‐for‐use method strategy is also presented to enable an optimum allotment of resources for bile related analysis; and finally, the use of bile matrix in several mechanistic studies to probe efflux mechanisms and/or drug–drug interaction potential has been presented with relevant case studies. Copyright © 2010 John Wiley & Sons, Ltd.