The global analysis of metabolism by liquid chromatography coupled to mass spectrometry is often hampered by a large amount of biological and technical variability. Here, we introduce an experimental and analytical strategy that can produce robust metabolome profiles in the face of this challenge. By applying a new computational approach based on concordance analysis to an extremely large number of analytical replicates, we are able to show that the overexpression of an antisense non-coding RNA targeting glutamine synthetase I results in a major reorganization of the metabolism of Streptomyces coelicolor, the model species of antibiotic-producing bacteria. We identified 97 metabolites with statistically significant reproducible dynamic behavior across the time series. The observed metabolic changes are very rapid, specific and widespread across metabolism, but focus on the nitrogen assimilation pathways. Our results demonstrate the power of highly replicated experimental designs for the robust characterization of metabolite dynamics. The identified global rearrangement of metabolism suggests the usefulness of RNA interference as an efficient strategy to manipulate the physiology of bacteria with wider biotechnological applicability in microorganisms.