Abstract A mass spectrometry based method for the direct determination of kinetic constants for phosphoglucose isomerase (PGI) and phosphomannose isomerase (PMI) is described. PGI catalyzes the interconversion between glucose-6-phosphate (Glc6P) and fructose-6-phosphate (Fru6P) and PMI performs the same function between mannose-6-phosphate (Man6P) and Fru6P. These two enzymes are essential in the pathways of glycolytic or oxidative metabolism of carbohydrates and have been considered as potential therapeutic targets. Traditionally, they are assayed either by spectrophotometric detection of Glc6P with one or more coupling enzymes or by a colorimetric detection of Fru6P. However, no suitable assay for Man6P has been developed yet to study the reaction of PMI in the direction from Fru6P to Man6P. In the work presented herein, a general assay for the isomeric substrate-product pair between Glc6P and Fru6P or between Man6P and Fru6P was developed, with the aim of directly studying the kinetics of PGI and PMI in both directions. The 6-phosphorylated aldose and ketose isomers were distinguished based on their corresponding tandem mass spectra (MS 2 ) obtained on a quadrupole ion trap mass spectrometer, and a multicomponent quantification method was utilized to determine the composition of binary mixtures. Using this method, the conversion between Fru6P and Glc6P and that between Fru6P and Man6P are directly monitored. The equilibrium constants for the reversible reactions catalyzed by PGI and PMI are measured to be 0.3 and 1.1, respectively, and the kinetic parameters for both substrates of PGI and PMI are also determined. The values of K M and V max for Fru6P as substrate of PMI are reported to be 0.15 mM and 7.78 μmol/(min mg), respectively. All other kinetic parameters measured correlate well with those obtained using traditional methods, demonstrating the accuracy and reliability of this assay.