Abstract: Breast cancer is the most frequently diagnosed cancer in women and a major cause of cancer-related deaths. Among its subtypes, triple-negative breast cancer (TNBC), which is defined by the absence of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2) expression, is particularly aggressive and tends to grow and spread fast. Doxorubicin (DOX) is widely used in clinical chemotherapy for TNBC. Studies have shown that this anticancer drug takes effect by inserting into DNA strands, enhancing free radical production, and causing oxidative damage to mitochondria. However, its molecular mechanism remains incompletely understood. Single-cell metabolomics has advanced rapidly in recent years, emerging as a powerful tool for studying tumor heterogeneity and disease mechanisms. In comparison to bulk analysis, single-cell metabolomics minimizes metabolite alterations introduced by sample preparation and better captures the actual metabolic state of cells. Studying the metabolic effects of DOX at the single-cell level helps clarify its anti-tumor mechanisms. In this study, a nanocapillary-based electrospray ionization mass spectrometry (ESI-MS) technique was used to explore the mechanism of DOX in single MDA-MB-231 cancer cells. This capillary-based method enables in situ sampling and metabolite identification in single living cells. Orthogonal partial least squares discriminant analysis (OPLS-DA) of the control group and DOX-treated MDA-MB-231 cells revealed a significant distinction between the metabolic profiles of DOX-treated and control groups, including 20 differential metabolites with variable importance in the projection (VIP) values greater than 1. Moreover, the heatmap demonstrated strong clustering within each group and a clear separation between the DOX-treated and control groups. Pathway enrichment analysis was performed on the differential metabolites to explore their association with anti-tumor mechanisms. Among them, the upregulation of 5-deoxyribose-1-phosphate is consistent with the mechanism of DOX-induced DNA damage. In addition, alanine, aspartate, and glutamate metabolism pathways were significantly enriched, which suggests that DOX affects tumor cells not only through causing DNA damage, but also by disrupting amino acid metabolism. In summary, this work reveals the antitumor mechanism of DOX from a single-cell perspective. The information obtained can effectively reveal drug mechanisms at the single-cell scale and may support more accurate cancer treatment strategies. It can also provide a technical reference for further studies on tumor metabolism. Subsequent studies may further extend the application of this technique in real tumor cell growth environments.