Fluorescent conjugates of drugs can be used to study cellular functions and pharmacology. These compounds interact with proteins as substrates or inhibitors, helping in the development of unique fluorescence-based methods to study in vivo localization and molecular mechanisms. P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette (ABC) transporter that effluxes most anticancer drugs from cells, contributing to the development of drug resistance. To study the transport function of P-gp, we synthesized a Bodipy-labeled fluorescent conjugate of cyclosporine A (BD-CsA). After synthesis and characterization of its chemical purity, BD-CsA was compared with the commonly used 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-CsA probe. In flow cytometry assays, the fluorescence intensity of BD-CsA was almost 10 times greater than that of NBD-CsA, enabling us to use significantly lower concentrations of BD-CsA to achieve the same fluorescence levels. We found that BD-CsA is recognized as a transport substrate by both human and mouse P-gp. The rate of efflux of BD-CsA by human P-gp is comparable to that of NBD-CsA. The transport of BD-CsA was inhibited by tariquidar, with similar IC50 values to those for NBD-CsA. BD-CsA and NBD-CsA both partially inhibited the ATPase activity of P-gp with similar IC50 values. In silico docking of BD-CsA and NBD-CsA to the human P-gp structure indicates that they both bind in the drug-binding pocket with similar docking scores and possibly interact with similar residues. Thus, we demonstrate that BD-CsA is a sensitive fluorescent substrate of P-gp that can be used to efficiently study the transporter's localization and function in vitro and in vivo. SIGNIFICANCE STATEMENT: The goal of this study was to develop an effective probe to study drug transport by P-glycoprotein (P-gp). Fluorophore-conjugated substrates are useful to study the P-gp transport mechanism, structural characteristics, and development of its inhibitors. Cyclosporine A (CsA), a cyclic peptide comprising 11 amino acids, is a known substrate of P-gp. P-gp affects CsA pharmacokinetics and interactions with other coadministered drugs, especially during transplant surgeries and treatment of autoimmune disorders, when CsA is given as an immunosuppressive agent. We synthesized and characterized Bodipy-FL-CsA as an avid fluorescent substrate that can be used to study the function of P-gp both in vitro and in vivo. We demonstrate that Bodipy-FL-conjugation does not affect the properties of CsA as a P-gp substrate.