Metabolic analysis of single cells to uncover cellular heterogeneity and metabolic noise is limited by the available tools. In this study, we demonstrate the utility of capillary microsampling electrospray ionization mass spectrometry with ion mobility separation for nontargeted analysis of single cells. On the basis of accurate mass measurements and collision cross-section determination, a large number of chemical species, 22 metabolites and 54 lipids, were identified. To assess the cellular response to metabolic modulators, the adenylate energy charge (AEC) levels for control and rotenone treated cells were evaluated. A significant reduction in the AEC values was observed for rotenone treated cells. For the cells under oxidative stress, the mean value for the [reduced glutathione (GSH)]/[oxidized glutathione (GSSG)] ratio was significantly decreased, whereas the distribution of the [uridine diphosphate N-acetylhexosamine (UDP-HexNAc)]/[uridine diphosphate hexose (UDP-hexose)] ratio exhibited dramatic tailing to higher values. Lipid turnover rates were studied by pulse-chase experiments at the single cell level.