OATPs are exchangers mediating the cellular uptake of hormones, bilirubin, bile acids and various drugs, including antivirals, chemotherapeutics, antibiotics and statins. OATP1B1 and OATP1B3 participate in hepatic elimination of their drug substrates, while OATP2B1 is the most abundant OATP in the intestine. Inhibition of their transport activity e.g., by co-administration of their drug or food component substrates may alter pharmacokinetics or even result in toxicity. Hence the study of hepatic OATPs is recommended during drug development (FDA, EMA and PMDA recommendations). OATP drug interactions are generally measured using radiolabeled substrates. However, the use of fluorescent compounds as test substrates of OATPs (and other transporters) provides a more sensitive tool1,2. Recently we identified Cascade Blue hydrazide (CB) and Alexa Fluor 405 (AF405) pyranine-based dyes as transported substrates of OATPs, applicable in drug interaction tests3. The aim of the current study was to analyze the transport of further pyranine analogs with the aim to map substrate interaction profile of hepatic OATPs, and to find more affordable OATP test substrates4. Transport of the fluorescent dyes was measured in adherent cell lines stably overexpressing OATP1B1, 1B3 or 2B1. Positive hit dyes were further characterized to find optimal assay conditions. The assay based on best performing dyes was validated by measuring uptake in the presence of documented OATP inhibitors. Finally, using the novel assay interaction between a set of flavonoids and OATPs was determined5,6. The developed fluoresce-based assay is a sensitive tool allowing large scale screen of OATP inhibitors and substrates.