Objective: To evaluate the effect of a thiolated polymer lubricant, chitosan-N-acetylcysteine conjugate (CNAC), in a mouse model of dry eye. Methods: Eye drops containing 0.5% C-NAC, 0.3% CNAC, a vehicle ( control group), artificial tears, or fluorometholone were applied in a masked fashion in a mouse model of induced dry eye from 3 days to 4 weeks after botulinum toxin B injection. Corneal fluorescein staining was periodically recorded. Real-time reverse transcriptase-polymerase chain reaction and immunofluorescence staining were performed at the end of the study to evaluate inflammatory cytokine expressions. Results: Mice treated with C-NAC, 0.5%, and fluorometholone showed a downward trend that was not statistically significant in corneal staining compared with the other groups. Chitosan-NAC formulations, fluorometholone, and artificial tears significantly decreased IL-1 beta (interleukin 1 beta), IL-10, IL-12 alpha, and tumor necrosis factor alpha expression in ocular surface tissues. Conclusions: The botulinum toxin B-induced dry eye mouse model is potentially useful in evaluating new dry eye treatment. Evaluation of important molecular bio-markers suggests that C-NAC may impart some protective ocular surface properties. However, clinical data did not indicate statistically significant improvement of tear production and corneal staining in any of the groups tested.