Several bile acid (BA) transporters are involved in the enterohepatic BA circulation between the liver and gut, including the hepatic Na+/taurocholate cotransporting polypeptide (NTCP) and the intestinal apical sodium-dependent BA transporter (ASBT). Fluorescent BA derivatives are helpful to measure and visualize BA transport in vitro and in vivo. We used 4-nitrobenzo-2-oxa-1,3-diazole (NBD) as the labeling fluorophore and synthesized a series of 3-NBD-coupled BA. While 3α-NBD-taurocholic acid, 3β-NBD-taurocholic acid, 3α-NBD-glycocholic acid, and 3β-NBD-glycocholic acid showed significant transport rates for human NTCP, mouse mNtcp, and mouse mAsbt, human ASBT only showed reliable transport activity for 3α-NBD-glycocholic acid. In general, NBD coupling to the 3α-position proved superior to the 3β-position, and the NBD-BA with glycine conjugation exhibited the highest overall transport rates. None of the synthesized NBD-BA was transported by the organic anion transporting polypeptides OATP1B1 and OATP1B3. Overall, 3α-NBD-glycocholic acid is most appropriate for fluorescence-based transport assays to evaluate NTCP and ASBT inhibitors.