The mucus lining of the gastrointestinal tract epithelium is recognized as a barrier to efficient oral drug delivery. Recently, a new in vitro model for assessment of drug permeation across intestinal mucosa was established by applying a biosimilar mucus matrix to the surface of Caco-2 cell monolayers. The aim of the present study was to gain more insight into the steric and interactive barrier properties of intestinal mucus by studying the permeation of peptides and model compounds across the biosimilar mucus as well as across porcine intestinal mucus (PIM). As PIM disrupted the Caco-2 cell monolayers, a cell-free mucus barrier model was implemented in the studies. Both the biosimilar mucus and the PIM reduced the permeation of the selected peptide drugs to varying degrees illustrating the interactive properties of both mucus matrices. The reduction in peptide permeation was decreased depending on the cationicity and H-bonding capacity of the permeant clearly demonstrated by using the biosimilar mucus, whereas the larger inter sample variation of the PIM matrix obstructed similarly clear conclusions. Thus, for mechanistic studies of permeation across mucus and mucosa the biosimilar mucus offers a relevant and reproducible alternative to native mucus.