The 13C-labeling technique is a powerful characterization tool within the field of metabolic engineering aimed at determining intracellular steady state fluxes. The provided metabolic snapshots enable researchers to better understand and predict the phenotypic behavior of a micro-organism as a result of genetic alterations and/or different environmental conditions. In general, two main methods can be distinguished for deriving metabolic flux patterns from measured 13C-label distributions; (i) the local flux analysis approach, which determines the intracellular fluxes around a selected metabolite node and (ii) the whole isotopomer modeling approach which aims at estimating all fluxes throughout a predefined reaction network model. In this thesis the different 13C-labelling techniques were further developed and the available analytical platforms were applied for the analysis of the metabolic fluxes in the well-characterized yeast Saccharomyces cerevisiae and the less-studied filamentous fungi Penicillium chrysogenum. Throughout this thesis special attention was paid to the flux through the oxidative branch of the pentose phosphate pathway (PPP), which is of prime importance for penicillin synthesis in P. chrysogenum.