First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113-7D using liquid chromatography - electrospray ionization MS/MS (LC-ESI-MS/MS). A glucose-limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (μ) = 0.05 h^-1 in a medium containing only naturally labeled (99% U-¹²C, 1% U- ¹³C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) ¹³C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h ∼80% of the measured glycolytic metabolites were U-¹³C-labeled. Surprisingly, during the next 3 h no significant increase of the U-¹³C-labeled metabolites occurred. Second, we demonstrate for the first time the LC-ESI-MS/MS-based quantification of intracellular metabolite concentrations using U-¹³C-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U-¹³C-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U-¹³C Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U-¹³C-labeled equivalent, thereby eliminating drawbacks of LC-ESI-MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U- ¹³C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for.