Recent studies suggest that short chain aliphatic beta-nitro alcohols can cross-link corneoscleral tissue under physiologic conditions. The present study was conducted to determine the cytotoxic threshold for these agents in vitro and to draw comparisons to commonly used topical ophthalmic agents. Primary cultures of bovine corneal endothelial cells were grown to confluence in 24- and 96-well plates using standard protocol. The cells were exposed to three beta-nitro alcohols, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-2-pentanol in a range of concentrations from 0.1 to 10 mM. After a 48-hour exposure, cell necrosis and apoptosis were evaluated using trypan blue, propidium iodide, and annexin V staining. In addition, a review of the ophthalmic literature was conducted to derive comparisons with agents commonly used in clinical practice. An all-or-none response was observed for each compound. Positive staining with trypan blue, propidium iodide, and annexin V occurred at identical concentrations. The most toxic of the group was 2-nitro-1-propanol. The cytotoxic level for 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-2-pentanol was 3 mM (0.0273%), 1 mM (0.0105%), and 3 mM (0.0399%), respectively. Furthermore, by comparison with several agents used in ophthalmic practice such as fluoroquinolone antibiotics, anti-proliferative agents, and benzalkonium chloride, the beta-nitro alcohols exhibit less toxicity in vitro. Short chain aliphatic beta-nitro alcohols exhibit favorable in vitro toxicity thresholds. The result of this study encourages further evaluation of these compounds as potential pharmacologic topical stiffening agents for corneoscleral disorders.