The antibodies against Diphtheria toxin in human sera were measured by the ELISA method and the antitoxin titer was calculated using the parallel line assay method which is a quantitative bioassay method. The reproducibility of this method and the comparison of the titer between this method and cell culture method were proven. The dose response curve of standard antiserum and test serum calculated by the parallel line assay method showed linearity, and the regression line of the test serum was parallel to the standard serum. The coefficients of variance (CV) of the antitoxin titers obtained from triplicate measurements of 3 serum samples ranged from 9.1 to 36.0%. The serum at low titer gave a higher CV level. A good correlation was observed between the Diphtheria antitoxin titer by the neutralizing test in cultured cells and that measured by ELISA by the parallel line assay method. The coefficient of regression was 0.996 and the coefficient of the correlation was 0.899.