The gastrointestinal mucus is a hydrogel lining the luminal side of the gastrointestinal epithelium. Mucus is vital for gut homeostasis because it protects the epithelium from the noxious external environment. However, from a drug delivery perspective, drugs have to permeate through the mucus to reach the epithelium; therefore, mucus might pose a barrier to drug absorption. Most of the information about mucus derives from fundamental studies performed on rodents. However, information from larger preclinical animal species is highly warranted for improving study designs and guiding better interpretation of data from preclinical assessments. Furthermore, improved understanding of the nature of the gastrointestinal mucus would enable the development of in vitro mucus models with increased biorelevance. These could then be implemented in drug absorption assays to improve the (bio)predictability. Well-informed in vitro mucus models would enable quick and less variable screening of drug candidates in the early drug development stages. Finally, these models would contribute to reduction, refinement, and replacement (the 3Rs) of animal usage in the drug development process. This thesis aims to improve our understanding of the nature of gastrointestinal mucus and its impact on drug absorption. For this purpose, mucus from the complete gastrointestinal tract of pigs and dogs was characterized and the diffusion of physicochemically diverse FITC-dextrans through colonic mucus was studied, both ex vivo and in vitro. The characterization of the gastrointestinal mucus focused on properties relevant for drug absorption and revealed the physiological characteristics, composition, and structural profiles from the various gastrointestinal regions. The findings pointed towards substantial differences between small intestinal and colonic mucus in both species and served as the basis for developing artificial colonic mucus models for drug permeation assessments. Porcine and canine artificial mucus models were developed and validated against the respective native secretions in terms of structural properties and demonstrated their potential to capture the key diffusion patterns of FITC-dextrans observed in native colonic mucus. Overall, this work provided insights into key properties of mucus from large preclinical species and validated tools for the assessment of the impact of mucus on drug absorption
