Precise metabolic flux analysis (MFA) by gas chromatography-mass spectrometry (GC-MS) and computer calculation was performed, and the consistency of the estimated results was verified by independently performed nuclear magnetic resonance (NMR) analysis. The precise estimation of flux by the integration method of the mass isotopomer signal, defined as the coefficient of variance (CV) of multiple determination, was investigated, and the results estimated using different data sets with the same magnitude of error were confirmed. The CV of multiple determinations was sufficiently small to discuss and compare the fluxes of a metabolic pathway. The estimated fluxes using the GC-MS data were cross-validated with the NMR data that were independently measured and not used for MFA. The developed method was successfully applied to the MFA of the growth phase of two different glutamate-producing coryneform bacteria, Corynebacterium glutamicum and C. efficiens. The difference in the growth rate between these two bacterial species was discussed while considering the results of MFA, including forward and backward (exchange) fluxes.