Background Quantitative knowledge of intracellular fluxes is important for a comprehensive characterization of metabolic networks and their functional operation. In contrast to direct assessment of metabolite concentrations, in vivo metabolite fluxes must be inferred indirectly from measurable quantities in 13C experiments. The required experience, the complicated network models, large and heterogeneous data sets, and the time-consuming set-up of highly controlled experimental conditions largely restricted metabolic flux analysis to few expert groups. A conceptual simplification of flux analysis is the analytical determination of metabolic flux ratios exclusively from MS data, which can then be used in a second step to estimate absolute in vivo fluxes. Results Here we describe the user-friendly software package FiatFlux that supports flux analysis for non-expert users. In the first module, ratios of converging fluxes are automatically calculated from GC-MS-detected 13C-pattern in protein-bound amino acids. Predefined fragmentation patterns are automatically identified and appropriate statistical data treatment is based on the comparison of redundant information in the MS spectra. In the second module, absolute intracellular fluxes may be calculated by a 13C-constrained flux balancing procedure that combines experimentally determined fluxes in and out of the cell and the above flux ratios. The software is preconfigured to derive flux ratios and absolute in vivo fluxes from [1-13C] and [U-13C]glucose experiments and GC-MS analysis of amino acids for a variety of microorganisms. Conclusion FiatFlux is an intuitive tool for quantitative investigations of intracellular metabolism by users that are not familiar with numerical methods or isotopic tracer experiments. The aim of this open source software is to enable non-specialists to adapt the software to their specific scientific interests, including other 13C-substrates, labeling mixtures, and organisms.