Mucosal drug delivery accounts for various administration routes (i.e., oral, vaginal, ocular, pulmonary, etc.) and offers a vast surface for the permeation of drugs. However, the mucus layer which shields and lubricates all mucosal tissues can compromise drugs from reaching the epithelial site, thus affecting their absorption and therapeutic effect. Therefore, the effect of the mucus layer on drug absorption has to be evaluated early in the drug-development phase, prior to in vivo studies. For this reason, we developed a simple, cost-effective and reproducible method employing UV-visible localized spectroscopy for the assessment of the interaction between mucin and drugs with different physicochemical characteristics. The mucin-drug interaction was investigated by measuring the drug relative diffusivity (Drel) in the presence of mucin, and the method was validated by fitting experimental and mathematical data. In vitro permeability studies were also performed using the mucus-covered artificial permeation barrier (mucus-PVPA, Phospholipid Vesicle-based Permeation Assay) for comparison. The obtained results showed that the diffusion of drugs was hampered by the presence of mucin, especially at higher concentrations. This novel method proved to be suitable for the investigation on the extent of mucin-drug interaction and can be successfully used to assess the impact that the mucus layer has on drug absorption.