Introduction <p>Central carbon metabolism (CCM) is the primary metabolic hub of the cell, governing energy production and providing precursors essential for a myriad of biosynthetic pathways. Developing analytical tools that can identify and quantify intermediates of these metabolic reactions is crucial for studying cell metabolism in biomedical and biotechnological applications.</p> Objective <p>This study proposes a liquid chromatography (LC)-high-resolution (HR) mass spectrometry (MS) method, covering the CCM of mammalian cell systems.</p> Methods <p>Cells were extracted using a one-step liquid extraction, recovering the hydrophilic metabolites. A stable isotope dilution approach was employed, utilizing a U-¹³C-yeast internal standard (IS). A LC-HRMS metabolomics method using hydrophilic interaction liquid chromatography (HILIC) coupled to a Zeno-time-of-flight (ZenoTOF) MS was implemented for metabolite semi-quantification.</p> Results <p>A total of 82 CCM metabolites is reported, of which 77 were confirmed with authentic standards, and for 63 , linearity ranges were obtained. IS normalization enhanced overall robustness, from sample preparation to metabolite semi-quantification. To study the effects on CCM by 5 chemical inhibitors (2-deoxy-<span>d</span>-glucose, etomoxir, UK-5099, rotenone, and 3-nitropropionic acid), our HILIC-HR-TOF-MS method was used. The approach proved efficient in capturing altered metabolite concentrations, within implicated metabolic reactions, as a consequence of inhibitor exposure.</p> Conclusion <p>Our HILIC-HR-TOF-MS metabolome method is efficient in mapping changes in metabolic intermediates of the CCM in mammalian cells. This approach holds potential for analysing a variety of biological samples across a range of applications, from drug development to biomedicine.</p>