High-throughput molecular analysis has become an integral part in organismal systems biology. In contrast, due to a missing systematic linkage of the data with functional and predictive theoretical models of the underlying metabolic network the understanding of the resulting complex data sets is lacking far behind. Here, we present a biomathematical method addressing this problem by using metabolomics data for the inverse calculation of a biochemical Jacobian matrix, thereby linking computer-based genome-scale metabolic reconstruction and in vivo metabolic dynamics. The incongruity of metabolome coverage by typical metabolite profiling approaches and genome-scale metabolic reconstruction was solved by the design of superpathways to define a metabolic interaction matrix. A differential biochemical Jacobian was calculated using an approach which links this metabolic interaction matrix and the covariance of metabolomics data satisfying a Lyapunov equation. The predictions of the differential Jacobian from real metabolomic data were found to be correct by testing the corresponding enzymatic activities. Moreover it is demonstrated that the predictions of the biochemical Jacobian matrix allow for the design of parameter optimization strategies for ODE-based kinetic models of the system. The presented concept combines dynamic modelling strategies with large-scale steady state profiling approaches without the explicit knowledge of individual kinetic parameters. In summary, the presented strategy allows for the identification of regulatory key processes in the biochemical network directly from metabolomics data and is a fundamental achievement for the functional interpretation of metabolomics data.