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In the past decades, drug metabolizing enzymes (DME) and their role in drug interaction have been intensively investigated. Apart from DME, transporters are also involved in the absorption, distribution, and excretion of drugs; this implies that (pharmacokinetic) drug interactions may be mediated by transporters as well. However a less systematic approach is used for transporter-related drug elimination and interactions. This can be attributed to the fact that preclinical model systems to study transporter-based drug interactions differ from those typically used for metabolism- based interactions. In addition specific inhibitors and substrates are often lacking for transporters. In this research, we focused on characterizing a fluorescent transporter substrate (fluorescent bile salt analogue) with respect to its in vitro hepatobiliary disposition in various in vitro models. In addition, the utility of this probe substrate for assessment of hepatic transporter mediated drug interactions by using HIV Protease inhibitor as model interacting compounds was also investigated.