Relative Expression Factors (REFs) are used to scale in vitro transporter kinetic data via In Vitro–In Vivo Extrapolation linked to Physiologically-Based Pharmacokinetic (IVIVE-PBPK) models to clinical observations. Primarily two techniques to quantify transporter protein expression are available, immunoblotting and LC-MS/MS. Literature collated Relative Expression Factors (REF) ranged from 0.4 to 5.1 and 1.1 to 90 for intestinal Pglycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), respectively. The impact of using human jejunum-Caco-2 REFs, for P-gp (REFiP-gp) and BCRP (REFiBCRP), generated from the same samples, employing different proteomic methodologies from independent laboratories, on PBPK outcomes was assessed. A 5-fold decrease in REFiP-gp for a single oral dose of digoxin resulted in a 1.19- and 1.31-fold higher plasma AUC and Cmax, respectively. All generated REFiP-gp values led to simulated digoxin Cmax values within observed ranges; however, combining kinetic data generated from a different laboratory, with the 5-fold lower REFiP-gp, could not recover a digoxin-rifampicin DDI; emphasising the necessity to obtain transporter-specific kinetic estimates and REFs from the same in vitro system. For a theoretical BCRP compound, with absorption taking place primarily in the jejunum, a decrease in the REFiBCRP from 2.22 (University of Manchester) to 1.11 (Bertin Pharma) promoted proximal intestinal absorption, while delaying tmax 1.44-fold. Laboratory-specific differences in REF may lead to different IVIVE-PBPK outcomes. To understand the mechanisms underlying projected PK liabilities, it is important to assess the potential impact of bias on the generation of REFs on an inter-individual basis within a target population.