Single-cell analysis of metabolites by mass spectrometry (MS) is challenging due to the very limited volume and inherent molecular complexity of the sample. Quantitative metabolomic analysis of individual cells provides information on the metabolic heterogeneity of cells unattainable by aggregate analysis of multiple cells. Depending on the ionization method, MS can offer quantitative analysis for a broad class of metabolites exhibiting both high sensitivity and selectivity. Laser ablation electrospray ionization (LAESI) has been successfully exploited to analyze metabolites from broad range of biological samples, including single cells and small cell populations. In this work, we describe a protocol for the relative quantitation of metabolites in single cells by LAESI-mass spectrometry.