One of the most common mechanisms of hepatotoxicity is drug-induced cholestasis. Hence, new approaches for screening the cholestatic potential of drug candidates are desirable. In this context, we describe herein the use of synthetic 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorescent conjugates of cholic acid (ChA) at positions 3alpha, 3beta, 7alpha, and 7beta for in vitro assessment of bile acid uptake. All the conjugates show a strong absorption band between 400 and 550 nm and have a fluorescence quantum yield of approximately 0.45, with an emission maximum centered at approximately 530 nm. After their photophysical characterization, 3alpha-, 3beta-, 7alpha-, and 7beta-NBD-ChA were used to monitor uptake in freshly isolated rat hepatocytes by means of a previously optimized flow cytometry technique. Transport of the cholic acid derivatives inside the cell was detected and quantified by measuring the increase of NBD green fluorescence within cells over time. The effect of troglitazone, a well-known inhibitor of bile acid uptake by the sodium taurocholate co-transporting polypeptide, supports the specificity of fluorescent NBD-ChA transport. According to the final intracellular fluorescence level attained and the uptake rate, 3alpha-NBD-ChA was found to be the most efficient derivative. Furthermore, sodium valproate, cyclosporin A, and chlorpromazine decreased the uptake of 3alpha-NBD-ChA, in agreement with their relative in vivo potency as cholestatic compounds; in contrast, sodium citrate (the negative control) had no effect. These results support the suitability of the in vitro flow cytometric assay with NBD-ChA to detect compounds that affect bile acid uptake.