The efflux proteins P-glycoprotein (P-gp), BCRP and members of the MRP-family (MRPs) are increasingly recognized as determinants of the absorption, tissue distribution and excretion of numerous drugs. A widely applied in vitro screening method, to assess the effect of these efflux transporters in transmembrane transport of drugs is based on the use of peripheral blood mononuclear cells (PBMC), in which the efflux of fluorescent dye Rhodamine 123 (Rh-123) can be easily measured. In avian species, the isolation of PBMCs is compromised by the presence of thrombocytes having approximately the same size. As an alternative, we validated the use of isolated splenocytes to assess Rhodamine 123 transport in the presence and absence of specific inhibitors for P-gp, MRPs and BCRP. Rh-123 efflux was concentration-dependent with the percentage of efflux that decreased with increasing concentrations. P-gp inhibitors, PSC833 and GF120918, significantly inhibit Rh-123 efflux, whereas inhibitors for MRPs and BCRP, MK571 and Ko-143, respectively, have a limited inhibitory effect. However, the effect of GF120918 was more pronounced as compared to PSC833, suggesting an additional role for BCRP next to P-gp in Rh-123 efflux. Moreover, fluoroquinolones were selected to test the applicability of the described model. None of these fluoroquinolones significantly inhibit P-gp function at concentrations up to 50 μM, with exception of danofloxacin and danofloxacin mesylate that were found to reduce Rh-123 efflux by approximately 15%.