Sustained drug delivery to mucosal surfaces has the potential to improve the effectiveness of prophylactic and therapeutic treatments for numerous diseases and conditions, including inflammatory bowel disease, sexually transmitted diseases, cystic fibrosis, glaucoma, dry eye, and various cancers. Sustained delivery systems such as nanoparticles can be useful for mucosal delivery, but recent work suggests they must penetrate the rapidly cleared mucus barrier that overlies all mucosal epithelia to achieve uniform distribution on epithelial surfaces and enhanced residence time. Thus, it is important to evaluate the mucus-penetrating ability of nanosized delivery systems in preclinical animal studies, and for administration to humans. We describe a simple ex vivo method to visualize and quantify nanoparticle transport in mucus on fresh mucosal tissues. Using this method in murine models, we observed variations in the mucus mesh at different anatomical locations, as well as cyclical variations that may have implications for mucosal delivery.