Abstract Current 13 C-metabolic flux analysis methods were reviewed as well as the weakness of the conventional metabolic flux analysis without 13 C-labeled experiments. Although it has been recognized that 13 C-labeling technique is powerful in estimating the metabolic fluxes, and the program-based flux analysis is necessary, one may not be confident with the result obtained without experiences and exhaustive trial and errors in practice due to its black box nature. In the present article, we call attention to the importance of investigating the relationships between fluxes and isotopomer or mass isotopomer distributions to understand the mechanism of generating specific isotopomers. Then, the experimental design for the preferred mixture of the specific 13 C-labeled substrate was discussed. The effect of the reversibility in the bidirectional flux on the isotopomer distribution was also mentioned, and it was shown why the reliability of the bidirectional fluxes becomes lower. Moreover, by noting that recent development of measurement techniques enables us to measure the isotopomer patterns of intracellular metabolites instead of proteinogenic amino acids, it is mentioned that this enables us to estimate the flux changes during time-variant batch culture. Some future perspectives are discussed in relation to the integration of different levels of information in the cell.