We report an absorption-based hyperspectral imaging and analysis technique to resolve unique physicochemical characteristics of subcellular substances in single erythrocytes. We constructed a microscope system installed with a spectral light engine capable of controlling the spectral shape of the illumination light by a digital micromirror device. The hyperspectral imaging system and the sequential maximum angle convex cone algorithm allow us to extract unique spectral signatures (i.e., endmembers) for different types of hemoglobin, such as oxyhemoglobin, methemoglobin, and hemozoin, and scatter from cell membrane in single erythrocytes. Further statistical endmember analysis, conducted on the hyperspectral image data, provides the abundances of specific endmembers, which can be used to build intracellular maps of the distribution of substances of interest. In addition, we perform modeling based on Mie theory to explain the scattering signatures as a function of scattering angle. The developed imaging and analysis technique enables label-free molecular imaging of endogenous biomarkers in single erythrocytes in order to build oxymetric standards on a cellular level and ultimately for in vivo as well.