Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions.