The major by-products in ethanol fermentatiion by Saccharomyces cerevisiae are glycerol(1.80% of glucose), acetaldehyde(1.50%), acetic acid(1.10%), lactic acid(0.13%) and formic acid(0.10%). The effects of glycerol, acetaldehyde and acetic acid which are produced comparatively in large amounts were studied for the development of a distillatiion process. By-products had nearly no effects on the vapor-liquid equilibrium and the theoretical number of plate. But the distillation columns having 10 and 11 theoretical number of plates were required in the case of acetic acid and acetaldehyde separation, respectively, to meet the specifica-tions for commercial fuel ethanol.