Current (13)C labeling experiments for metabolic flux analysis (MFA) are mostly limited by either the requirement of isotopic steady state or the extremely high computational effort due to the size and complexity of large metabolic networks. The presented novel approach circumvents these limitations by applying the isotopic non-stationary approach to a local metabolic network. The procedure is demonstrated in a study of the pentose phosphate pathway (PPP) split-ratio of Penicillium chrysogenum in a penicillin-G producing chemostat-culture grown aerobically at a dilution rate of 0.06h(-1) on glucose, using a tracer amount of uniformly labeled [U-(13)C(6)] gluconate. The rate of labeling inflow can be controlled by using different cell densities and/or different fractions of the labeled tracer in the feed. Due to the simplicity of the local metabolic network structure around the 6-phosphogluconate (6pg) node, only three metabolites need to be measured for the pool size and isotopomer distribution. Furthermore, the mathematical modeling of isotopomer distributions for the flux estimation has been reduced from large scale differential equations to algebraic equations. Under the studied cultivation condition, the estimated split-ratio (41.2+/-0.6%) using the novel approach, shows statistically no difference with the split-ratio obtained from the originally proposed isotopic stationary gluconate tracing method.