Calcein acetoxymethyl ester (calcein-AM) is a non-fluorescent dye that easily crosses cell membranes. Once in the cells, calcein-AM is transformed into the fluorescent calcein that is retained in the cytosol. However, calcein-AM is highly extruded by P-gpexpressing cells, thus reducing the intracellular fluorescence intensity . When coadministered with a P-gp inhibitor, the intracellular accumulation of the fluorescent calcein is increased and the increase in fluorescence intensity can be used as a measure of P-gp inhibition. Caco-2 cells (5 x 10 5 cells/well) were seeded into 96-well plates and incubated overnight. Next, cells were preincubated for 30 min with the selected compounds (10 µM) and then treated with calcein-AM (0.25 µM, Sigma-Aldrich). Zosuquidar (10 µM, Sigma-Aldrich) was used as a reference drug. After 30 min incubation at 37 °C, cells were washed with PBS and fluorescence was measured at 490 nm excitation 520 nm emission using a Victor Nivo multimode plate reader . Experiments were performed in triplicate.