Abstract: Citrate (CA) and isocitrate (ICA) are key isomeric metabolites in the tricarboxylic acid (TCA) cycle, synthesized sequentially from acetyl-CoA and oxaloacetate, with isomerization catalyzed by aconitase 2 (ACO2). The dynamic balance between CA and ICA is essential for cellular energy metabolism, redox homeostasis, and signaling. Understanding their distribution and regulation at the single-cell level is critical for deciphering cellular heterogeneity in physiological and pathological processes. However, conventional methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS), rely on chromatographic separation and necessitate metabolite extraction from large cell populations (＞1 000 cells), obscuring single-cell heterogeneity. Therefore, there is an urgent need for analytical methods capable of direct differentiation of CA and ICA within individual cells. Nano-electrospray ionization mass spectrometry (nanoESI-MS), known for its high sensitivity, low sample consumption, and minimal sample preparation, is well-suited for single-cell metabolomics. Although tandem mass spectrometry (MS/MS) can theoretically differentiate isomers via characteristic fragment ions, practical application in single cells is hindered by matrix suppression. To overcome this problem, an induced nano-electrospray ionization (InESI) technique was employed. InESI utilizes specific frequency alternating current pulses applied to an electrode, inducing ionization from the sample capillary without direct contact, effectively reducing salt interference and improving ionization efficiency and sensitivity. In this study, an InESI MS/MS method was established for the direct differentiation and quantitation of CA and ICA within single living cells. Analysis of standard solutions identified diagnostic fragment ions: m/z 87 for CA and m/z 117 for ICA. A relative quantitation calibration curve based on the intensity ratio (I₈₇/I₁₁₇) versus concentration ratio demonstrated excellent linearity (R²=0.993 0). Additionally, an absolute quantification curve for CA was constructed using the intensity of the precursor ion (m/z 191) in artificial intracellular solution. Applied to single HepG2 cells, the method successfully acquired full scan spectra (detecting endogenous metabolites such as glutamate (m/z 146) and glutathione (m/z 306) and targeted MS/MS spectra of the precursor ion (m/z 191, M−H⁻ for both isomers), enabling direct, label-free differentiation. Based on the established curves, intracellular concentrations of CA and ICA were determined to be approximately 205 μmol/L and 36 μmol/L, respectively. Further, exogenous incubation experiments (2 mmol/L CA, 2 mmol/L ICA, or 1 mmol/L CA+1 mmol/L ICA) revealed significant differences in the intracellular CA/ICA relative content ratio (6.27±1.19, 1.49±0.09, and 3.99±1.29, respectively), aligning with theoretical expectations and validating the method's reliability. This work provides a powerful tool for the rapid, direct differentiation and quantitation of CA and ICA isomers in single cells, offering a strategy for analyzing other isomeric metabolites in single-cell metabolomics.