A method has been developed for rapid quantification of nine glycolytic intermediates using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS/MS) to monitor the metabolism of glucose during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is significantly less than traditional ion exchange liquid chromatography assays or enzymatic assays. Complete glycolytic intermediate analysis by LC/MS/MS can be achieved in less than 7 min per sample. Quantification is accomplished using isotopically labeled glucose, glucose-6-phosphate, and pyruvate as internal standards. In addition, a method to deconvolute peak areas of coeluting structural isomers based on unique product ion ratios has been developed to allow accurate quantification of the individual isomers 2-phosphoglycerate and 3-phosphoglycerate, as well as glucose-6-phosphate and fructose-6-phosphate. Intrasample precisions for glycolytic intermediate measurements in cell-free extracts using this method vary between 0.9% and 11.8%, averaging 3.5% (RSD). Calibration curves are linear over the range 0.1-100 microg/mL, and detection limits are estimated at 2-49 ng/mL. Spike recoveries in cell extracts vary from 53% to 127% averaging 91%. This method has the potential to demonstrate correlation of glycolytic intermediate flux to microbial production profiles toward acceleration of the bioprocess development cycle.