MDR1, BCRP, and MRP2 transporter knockout cell lines are now available that enable identification of substrate/transporter interactions without chemical inhibitors or knockdown RNAi technology. The knockout cell lines are available as assay-ready plates or may be licensed. Each knockout cell line has its respective transporter(s) functionally knocked out. When conducting routine efflux ratio analyses with these cells, the absence of each respective transporter helps to determine substrate/transporter interactions unambiguously.