An online method was developed to monitor BCRP mediated efflux of fluorescent substrates in suspension cells. To this end, a 2-compartment system consisting of a transwell cup and a cuvette was used. In this system we were able to observe differences in efflux kinetics between BCRP overexpressing RPMI 8226/MR cells and parental myeloid RPMI 8226(s) cells using only 50,000 cells per experiment. 8226/MR cells displayed a larger cellular efflux rate of the BCRP substrate Hoechst 33342, as compared to the wildtype cells. This difference in efflux rate was completely decreased in the presence of the BCRP inhibitor Ko143.