Abstract: Drug resistance remains a major challenge in breast cancer chemotherapy, yet the metabolic alterations underlying this phenomenon are not fully understood. There is much evidence indicating the cellular heterogeneity among cancer cells, which exhibit varying degrees of metabolic reprogramming and thus may result in differential contributions to drug resistance. A home-built single-cell quantitative mass spectrometry (MS) platform, which integrates micromanipulation and electro-osmotic sampling, was developed to quantitatively profile the tricarboxylic acid (TCA) cycle metabolites at the single-cell level. Using this platform, the metabolic profiles of drug-sensitive MCF-7 breast cancer cells and their drug-resistant derivative MCF-7/ADR cells were compared. This results revealed a selective upregulation of downstream TCA cycle metabolites including α-ketoglutarate, succinate, fumarate, and malate in drug-resistant cancer cells, while early TCA metabolites remained largely unchanged. Furthermore, notable variations in the abundance of the metabolites were observed in individual cells. The comparative analysis also revealed that not all MCF-7/ADR cells exhibit the same degree of metabolic deviation from the parental line in the metabolites during resistance acquisition. The observed metabolic profiles indicate enhanced glutaminolysis, altered mitochondrial electron transport chain activity, and increased metabolic flexibility in drug-resistant cancer cells that support their survival under chemotherapeutic stress. The findings further suggest the potential for incorporating cellular metabolic heterogeneity into future drug resistance studies.