BACKGROUND: The In Vitro-In Vivo Extrapolation (IVIVE) of intestinal transporter-mediated drug clearance within Physiologically-Based Pharmacokinetic (PBPK) models has historically relied upon transporter expression data from relative quantification approaches (e.g. PCR or immunoblotting). Herein, an IVIVE strategy for scaling in vitro transporter kinetic data ‘per pmol of transporter’ expressed within a cell monolayer, via an individual’s absolute intestinal transporter abundance is described. The impact of scaling via an absolute approach for the P-glycoprotein (P-gp) probe substrate Digoxin is demonstrated in the Simcyp Simulator (V17R1, Sheffield, UK). METHODS: In vitro transporter kinetics i.e. Jmax and Km, or intrinsic transporter clearance (CLint,T) for efflux transporters are typically determined in Caco-2 or human transporter-transfected MDCK-II and LLC-PK1 monolayers, and can be corrected for the monolayers’ absolute transporter abundance(s) (pmol transporter) quantified via proteomic methods. To scale to the in vivo situation the following information is needed: 1) Region-specific transporter abundance for each intestinal segment (pmol/mg total membrane protein) obtained by literature meta-analysis. 2) An individual’s segmental transporter abundance is derived from the yield of Total Membrane Protein Per Intestine (TMePPI, 2737 ± 1807 mg, n=28) and Colon (TMePPC, 112 ± 37 mg, n=7) with segmental surface area normalisation to each individual. 3) An Inter-System Extrapolation Factor for Transporters (ISEF,T) is required to correct for in vitro-to-in vivo transporter activity differences. Multiplication of the region-specific abundance by the total membrane protein per intestinal segment, yields the individual’s segmental absolute transporter abundance (pmol transporter). To investigate the impact of transporter activity in vivo, the in vitro transporter kinetic data i.e. CLint,T,in vitro is scaled via the absolute transporter expression in each individual’s intestinal segment to provide a total segmental clearance (L/h). RESULTS & CONCLUSIONS: A verified Digoxin PBPK model using the relative scaling approach for intestinal P-gp was employed. The verified Digoxin model was modified to scale intestinal P-gp transport of Digoxin via the absolute approach by correcting the previously assigned P-gp Jmax of 434 pmol/min/cm2 by the absolute P-gp abundance per monolayer (0.84 pmol P-gp/21 day filter-grown Caco-2 monolayer), resulting in an intestinal P-gp Jmax of 517 pmol/min/pmol P-gp which was used alongside an ISEF,T of 1 in simulations. Healthy Volunteer simulations (10 trials of 10 individuals per trial, single oral dose of 0.5 mg) for Digoxin were performed in the Simcyp Simulator, where the proximal jejunum P-gp expression was 0.4 pmol/mg total membrane protein and region-specific P-gp expression was as previously described. The simulated Digoxin Cmax (2.69 ± 0.53 ng/mL) using the absolute approach compared favourably with the observed Cmax (range 1.39 – 3.0 ng/mL, n=10 independent studies, single oral dose 0.5 mg). This is the first reported study to describe and incorporate the IVIVE scaling of intestinal transport activity via absolute transporter abundances within a PBPK framework. The described absolute approach offers a fully mechanistic means to elucidate the importance of intestinal transporter activity on drug absorption.