A tetrazolium-based(MTT) assay was used to evaluate the viability of cultured porcine monolayer corneal endothelial cells. In this study, we tried to find out the best spectra at which scanning multiwell ELISA spectrophometer can obtain the widest range of absorbance (optical density, O.D.) and the ranges of numbers of corneal endothelial cells in which absorbance is in a significant linear relationship with cell numbers. Among the three tested spectra of 450, 540, and 630nm, the widest range of absorbance was at 540 nm, from 0.1193 to 0.6183. At all three tested spectra, numbers of corneal endothelial cells from 50 to 30,000 were in a significant linear relationship with absorbance. Therefore, in the future studies to assay the viability of corneal endothelial cells, we shall choose the spectrum of 540 nm for reading with a scanning multiwell ELISA spectrophotometer, and absorbance(O.D.) from 0.1193 to 0.6183 will be accepted, representing cell numbers from 50 to 30,000.