Absorption of drugs across any mucosal tissue may involve interactions with the mucus gel overlying the tissue. Drug binding to the mucus glycoproteins, in particular, can reduce the amount of free drug available for absorption. In order to evaluate the extent of drug binding to mucin, a purified model mucus system containing primarily the large glycoprotein fraction (400 kDa) of gastric mucus was developed for use in drug binding studies. The extent of binding of six selected compounds (albuterol, rifampicin, p-amino-salicylic acid, isoniazid, pyrazinamide, and pentamidine) to mucus glycoproteins was studied. The binding of each drug to a model plasma protein, bovine serum albumin (BSA), was also investigated. Binding studies were performed by diafiltration, which combines characteristics of equilibrium dialysis and ultrafiltration in a continuous system. All the compounds selected showed affinities of the same order of magnitude to mucin despite being chemically dissimilar and exhibiting differing ionization states. This suggests that binding to gastric mucus glycoproteins is non-specific in nature with similar types of binding forces involved in the binding of all the compounds tested. Results also showed that the drug-protein association constants for BSA and mucin were of the same order of magnitude only for drugs with low binding affinities. When the binding constants to BSA were moderate to high, the corresponding drug binding constants to mucin were lower by at least one order of magnitude. Based on these results, it can be concluded that the binding behavior of drugs to gastric mucin is non-specific in nature with binding constants of a low magnitude. BSA cannot be used to estimate binding to mucin, especially when the drug exhibits moderate to high affinity for BSA.