Madin-Darby canine kidney II (MDCKII) cells expressing one or several human transport proteins are commonly used for drug transport studies. A major drawback in these cell models is the expression of endogenous transporters such as canine multidrug resistance protein 1 (Mdr1/P-glycoprotein; Abcb1) and breast cancer resistance protein (Bcrp; Abcg2). Activity of these endogenous canine transport proteins can directly compete with the human transporters, which complicates interpretation of data generated from these studies and hence hinder the predictive power of these models in humans. To address these issues, we have generated three modified MDCKII cell lines: 1) MDCKII-BPnull is a double knockout (Mdr1-/-/Bcrp-/-), in which both the canine Mdr1 and Bcrp genes have been knocked out using CRISPR-Cas9 gene-editing technology. 2) MDR1-MDCKII-BPnull over expresses human MDR1 in this MDCKII-BP-null cell line, which was introduced as a piggyBac transposon.3) BCRP-MDCKII-BPnull over expresses human BCRP in the MDCKII-BPnull cell line, which was introduced as a piggyBac transposon. These cell lines demonstrate effective human transporters activity with no interference from canine Mdr1 and Bcrp transporters. The loss of canine Mdr1 and Bcrp in MDCKII-BP-null was confirmed by genome sequencing as well as transporter assays using 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)) as a substrate. Human MDR1 and BCRP activity in transfected cell lines were verified by Calcein-AM assay and Hoechst 33342 assay respectively. We expect the MDR1-MDCKII-BPnull and BCRP-MDCKII-BPnull cell lines are useful tools to identify human MDR1 and BCRP substrates as well as inhibitors. And the MDCKII-BP-null cell line will be an ideal host cell line for heterologous expression of other transporters of interest.