Viscoelastic mucus lines all mucosal surfaces of the body and forms a potential barrier to mucosal drug delivery. Mucus is mainly composed of water and mucins; high-molecular weight glycoproteins forming an entangled network. Consequently, mucus forms a steric barrier and due to its negative charge and hydrophobic domains, the overall hydrophilic mucus also presents an interactive barrier limiting the free diffusion of components within and through the mucus. Furthermore, mucus is a dynamic barrier due to its continuous secretion and shedding from the mucosal surfaces. Mucus is thus a highly complex gel barrier to drug delivery. Current knowledge of mucus characteristics and barrier properties, as achieved by state-of-the-art methodologies, is the topic of this MiniReview emphasizing the gastrointestinal mucus and an overall focus on oral drug delivery. Cell culture-based in vitro models are well-established as essential tools in drug research and development, but traditionally, mucus-containing models have only rarely been applied. However, a number of mucus-containing in vitro models have recently been described in the literature and their properties and applications will be reviewed and discussed. Finally, studies of peptide and protein drug diffusion in and through mucus and studies of mucus-penetrating nanoparticles are included to illustrate the mucus as a potentially important barrier to obtain sufficient bioavailability of orally administered drugs, and thus an important parameter to address in the development of future oral drug delivery systems. This article is protected by copyright. All rights reserved.