C-13-metabolic flux analysis (C-13-MFA) has become a key method for metabolic engineering and systems biology. In the most common methodology, fluxes are calculated by global isotopomer balancing and iterative fitting to stationary C-13-labeling data. This approach requires a closed carbon balance, long-lasting metabolic steady state, and the detection of C-13-patterns in a large number of metabolites. These restrictions mostly reduced the application of C-13-MFA to the central carbon metabolism of well-studied model organisms grown in minimal media with a single carbon source. Here we introduce non-stationary C-13-metabolic flux ratio analysis as a novel method for C-13-MFA to allow estimating local, relative fluxes from ultra-short C-13-labeling experiments and without the need for global isotopomer balancing. The approach relies on the acquisition of non-stationary C-13-labeling data exclusively for metabolites in the proximity of a node of converging fluxes and a local parameter estimation with a system of ordinary differential equations. We developed a generalized workflow that takes into account reaction types and the availability of mass spectrometric data on molecular ions or fragments for data processing, modeling, parameter and error estimation. We demonstrated the approach by analyzing three key nodes of converging fluxes in central metabolism of Bacillus subtilis. We obtained flux estimates that are in agreement with published results obtained from steady state experiments, but reduced the duration of the necessary C-13-labeling experiment to less than a minute. These results show that our strategy enables to formally estimate relative pathway fluxes on extremely short time scale, neglecting cellular carbon balancing. Hence this approach paves the road to targeted C-13-MFA in dynamic systems with multiple carbon sources and towards rich media. Biotechnol. Bioeng. 2013;110: 3164-3176. (c) 2013 Wiley Periodicals, Inc.