The central carbon metabolism is a major energy source of all living organisms. The alteration of metabolite levels in the central carbon metabolism can be used as a biomarker for different diseases. In many cases, the alteration can be observed only at the turnover rate of the metabolites, therefore to fully understand a mechanism underlying a phenotype, the alteration must be monitored not only metabolite levels but also metabolite turnover rates. Different enzymes regulate the metabolic processes in central carbon metabolism. The use of stable isotopes to track the changes in enzymes in central carbon metabolism can be utilized as a biomarker for various illnesses, including cancer. Therefore, targeted metabolomics or flux analysis with different stable isotopes of central carbon metabolism has been widely applied to identify the role of its in different diseases. The analysis of central carbon metabolism can be done with different analytical techniques; however, the unique advantage of GC-MS ensures priority in analysis because of its high resolution and analysis of most of the central carbon metabolites in a single run. We summarized recent development in central carbon metabolism study using GC-MS-based pathway targeted metabolomics and fluxomic analyses in this review. We focused on 18O-based stable isotope tracking for central carbon metabolism and compared its advantages and disadvantages to 13Cbased labeling studies.