AbstractEfflux transporters play an important role in drug absorption and also in multidrug resistance.ABCG2 (BCRP) is an efflux transporter conferring cross‐resistance to mitoxantrone (Mit), irinotecan (CPT11), and its active metaboliteSN38.MBLI87, a newABCG2 inhibitor has proven its efficacy againstABCG2‐mediated efflux in vitro and in vivo. This work aimed at modeling and quantifying the cellular interaction betweenMBLI87 and different substrates using a mechanistic template model. An in vitro competition experiment study was carried out withHEK293 cells overexpressingABCG2 exposed to fixed concentrations of substrates (Mit,CPT11,SN38) and toMBLI87 at several concentration levels. A nonlinear mixed‐effects transport inhibition model was developed to fit intracellular drug concentrations. In this model, drugs cross the cell membrane through passive diffusion, active drug efflux isABCG2 mediated, interaction between substrates and inhibitor occurs within the transporter. The interaction was found to be noncompetitive. TheMBLI87Ki was estimated to 141 nmforMit, 289 nmforCPT11, and 1160 nmforSN38. The ratio of intrinsic transport clearance divided by diffusion clearance was estimated to 2.5 for Mit, 1.01 forCPT11, and 5.4 forSN38. The maximal increase in the intracellular substrate concentration that is possible to achieve by inhibition of the transporter was estimated to 1.5 forMit, 0.1 forCPT11, and 4.4 forSN38. This mechanistic template model describes both drug accumulation and cellular transport, and the mixed‐effects approach allows an estimation of intra‐ and interassay variability. This model is of great interest to study cytotoxic cellular pharmacokinetics.