Glecaprevir and pibrentasvir are oral direct-acting antiviral agents approved in combination for treatment of chronic hepatitis C viral infection. In vitro studies identified the combination as potential clinically relevant inhibitors of the efflux transporters P glycoprotein (P-gp), breast cancer resistance protein (BCRP), and hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1/3. Glecaprevir inhibited P-gp, BCRP, OATP1B1, and OATP1B3 with IC50 values of 0.33, 2.3, 0.017, and 0.064 µM, respectively. Pibrentasvir inhibited P-gp, BCRP, and OATP1B1 with IC50 values of 0.036, 14, and 1.3 µM, respectively. Neither agent inhibited organic cation transporter (OCT) 1, OCT2, organic anion transporter (OAT) 1, OAT3, multidrug and toxin extrusion (MATE) 1, or MATE2K. Open-label Phase 1 clinical drug-drug interaction studies were conducted in healthy subjects to evaluate interaction potential of glecaprevir/pibrentasvir and coadministered selective substrates for P-gp (digoxin, dabigatran etexilate, sofosbuvir), BCRP (rosuvastatin, sofosbuvir), and OATP1B1/3 (pravastatin, rosuvastatin). Pharmacokinetic parameters maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) were evaluated for probe substrates alone and in combination with glecaprevir/pibrentasvir. Cmax central values increased by 72%, 105%, 123%, 462%, and 66% for digoxin, dabigatran, pravastatin, rosuvastatin, and sofosbuvir, respectively. AUC central values increased by 48%, 138%, 130%, 115%, and 125% for digoxin, dabigatran, pravastatin, rosuvastatin, and sofosbuvir, respectively. Exposure of sofosbuvir metabolite GS-331007 was similar with or without glecaprevir/pibrentasvir. Outcomes of the clinical drug-drug interaction studies confirmed clinically relevant inhibition of P-gp, BCRP, and OATP1B1/3, and were used to provide dosing guidance for the concomitant use of glecaprevir/pibrentasvir with relevant transporter substrates. SIGNIFICANCE STATEMENT N/A