Metabolic reprogramming in a subpopulation of cancer cells is a hallmark of tumor chemoresistance. However, single-cell metabolic profiling is difficult because of the lack of a method that can simultaneously detect multiple metabolites at the single-cell level. In this study, through hyperspectral stimulated Raman scattering (hSRS) imaging in the carbon-hydrogen (C–H) window and sparsity-driven hyperspectral image decomposition, we demonstrate a high-content hSRS (h ² SRS) imaging approach that enables the simultaneous mapping of five major biomolecules, including proteins, carbohydrates, fatty acids, cholesterol, and nucleic acids at the single-cell level. h ² SRS imaging of brain and pancreatic cancer cells under chemotherapy revealed acute and adapted chemotherapy-induced metabolic reprogramming and the unique metabolic features of chemoresistance. Our approach is expected to facilitate the discovery of therapeutic targets to combat chemoresistance. This study illustrates a high-content, label-free chemical imaging approach that measures metabolic profiles at the single-cell level and warrants further research on cellular metabolism.