Metabolomics has been a potential tool for strain improvement through analyzing metabolite changes in the context of different conditions. However, the availability of a universal metabolite profiling analysis is still a big challenge. In this study, we presented an optimized liquid chromatography-tandem mass spectrometry-based metabolomics methodology for Corynebacterium glutamicum, an important industrial workhorse. It was found that quenching the cellular metabolism with 5-fold volume of - 20 degrees C 40% methanol was highly recommended due to its lower cell damage rate and higher intracellular metabolite recovery rate. For extracting intracellular metabolites, ethanol/water (3:1, v/v) at 100 degrees C combined with acidic acetonitrile/water (1:1, v/v, with 0.1% formic acid) at - 20 degrees C achieved the unbiased metabolite profiling of C. glutamicum. The established methodology was then applied to investigate the intracellular metabolite differences between C. glutamicum ATCC 13032 and an mscCG-deleted mutant under biotin limitation condition. It was observed that in the presence of the functional l-glutamate exporter MscCG, biotin limitation led to accumulation of intracellular 2-oxoglutarate but not l-glutamate. Deletion of mscCG severely inhibited l-glutamate excretion and resulted in a dramatical increase of intracellular l-glutamate, which in turn affected the metabolite profile. The optimized metabolomics methodology holds promise for promoting studies on metabolic mechanism of C. glutamicum.