An high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method was established for the determination of 24 intracellular metabolites in E.coli.After quenching,washing and extraction,the samples were centrifuged to remove impurities,and then separated on a Waters Atlantis HILIC silica column.The mobile phases were acetonitrile and water(containing 10 mmol/L ammonium acetate) at a flow rate of 250 μL/min.The electrospray was operated in the negative ionization mode and the 24 metabolites were identified by multiple reaction monitoring(MRM) mode.The method of matrix-matched standard solution was adopted as the quantitative method.The calibration curves showed good linearity within the concentrations of 0.10~100 μmol/L with the correlation coefficients r >0.99.The limits of detection of the 24 metabolites were 0.05~0.34 μmol/L.The recoveries of the 24 metabolites were 83.7%~108.1% at the 3 spiked levels.The relative standard deviations were less than 10%.This method is simple,sensitive and accurate in the determination of 24 metabolites.