The aim of the present study was to investigate if mucus applied to Caco-2 cell monolayers protects cells from high concentrations of surfactants, while still allowing for an identification of the surfactant's inhibitory effects on P-glycoprotein (P-gp). Two types of porcine mucin and six surfactants (Polysorbate 20 (PS20) and 80 (PS80), Kolliphor EL (Kol. EL) and RH40 (Kol. RH40), Labrafil M 2125 CS (L.fil) and Labrasol (L.sol)) were applied to Caco-2 cells, and TEER, paracellular transport and P-gp mediated digoxin transport was measured. The results showed that 15% porcine mucin type II was incompatible with Caco-2 cell monolayer integrity, resulting in a dramatic drop in monolayer TEER and increased mannitol transport. In contrast, mucin type III was compatible with Caco-2 cell monolayers in the concentration range of 2.5 - 15% without substantially disturbing barrier properties. The highest concentration of mucin type III impaired the ability of all six surfactants to decrease P-gp mediated digoxin transport. Subsequently lowering the mucin concentration to 5% facilitated adequate protection of cells and enabled e.g., 5% PS20 to inhibit P-gp mediated digoxin transport. Overall, the present work is useful for early-stage permeability investigations on how mucus affects P-gp mediated transport in the presence of formulation excipients.