Sustained progress in metabolic engineering methodologies has stimulated new efforts toward optimizing fungal production strains such as through metabolite analysis of Penicillium chrysogenum industrial-scale processes. Accurate intracellular metabolite quantification requires sampling procedures that rapidly stop metabolism (quenching) and avoid metabolite loss via the cell membrane (leakage). When sampling protocols are validated, the quenching efficiency is generally not quantitatively assessed. For fungal metabolomics, quantitative biomass separation using centrifugation is a further challenge. In this study, P. chrysogenum intracellular metabolites were quantified directly from biomass extracts using automated sampling and fast filtration. A master/slave bioreactor concept was applied to provide industrial production conditions. Metabolic activity during sampling was monitored by 13C tracing. Enzyme activities were efficiently stopped and metabolite leakage was absent. This work provides a reliable method for P. chrysogenum metabolomics and will be an essential base for metabolic engineering of industrial processes.