Aldehyde dehydrogenase (ALDH) assays measure the accumulated fluorescence of enzyme products. However, cancer cells frequently co-express ALDH and ATP-binding cassette (ABC) transporters, which might mediate efflux of ALDH assay reagents. We demonstrate expression of active multidrug resistance protein1 (MDR1), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) in CT26 cancer cells as well as expression of MRP and BCRP in HT29 cancer cells. Without transporter inhibition, only small portions of both cell types were estimated to be ALDH-positive based on Aldefluor and AldeRed588 assays. However, MK-571 (MRP inhibitor) and novobiocin (BCRP inhibitor) substantially increased the rate of ALDH-positive CT26 cells based on either Aldefluor or AldeRed588 assays. Verapamil (MDR inhibitor) did not influence assay results. MK-571 also substantially increased the rate of ALDH-positive HT29 cells. Limiting dilution assays demonstrated greater numbers of tumor-spheres formed by Aldefluor-positive compared to -negative CT26 cells selected in the presence of MK-571 or novobiocin but not in their absence. These results reveal that Aldefluor and AldeRed588 products are efficient substrates for MRP- and BCRP-mediated efflux and substantially reduce estimated ALDH positivity rates in cancer cells. These findings demonstrate that complete blockade of these transporters is important to ensure accurate ALDH assay results and to develop newer assay techniques.