Abstract: A metabolic flux affecting substance production using cells is determined by 1) creating a stoichiometric matrix based on formulas of biochemical reactions from a substrate through a desired produced substance, 2) selecting the same number of independent metabolic fluxes from all metabolic fluxes as the degree of freedom of the stoichiometric matrix as free fluxes, 3) creating a sufficient number of random combinations of the free fluxes for a statistical analysis and calculating a metabolic flux distribution from each created combination based on the stoichiometric matrix, 4) obtaining a regression equation including a minimum number of free fluxes that shows a correlation with substance production from the calculated metabolic flux distributions by a multivariate statistical analysis, and 5) determining at least one metabolic flux affecting substance production based on a coefficient in the obtained regression equation.