Changes of metabolite concentrations in single cells are significant for exploring the dynamic regulation of important biological processes, such as cell development and differentiation. Accurate quantitation of metabolites is essential for single cell analysis. In this work, we proposed a quantitative method for single-cell metabolites by combining microwell array with droplet microextraction-mass spectrometry. The microwell can confine both single cells and extraction solvent in defined space, avoiding the irregular spread of trace internal standard solution during microextraction, which was the key to improve the precision and accuracy of quantification in extremely small-volume single-cell samples. Glucose-phosphate as a crucial metabolite in glycolysis was detected and quantified in single cells at this work. The calibration curve of glucose-phosphate was obtained with a linear range from amol (10-18 mol) to fmol (10-15 mol), providing the foundation of metabolite quantitation of single cells. We applied this method to investigate the changes of metabolites including glucose-phosphate, 2-deoxy-d-glucose-phosphate, and ribose-phosphate in single K562 cells stimulated by 2-deoxy-d-glucose. With the robust quantitative capabilities, the developed method holds great potential for studying a drugs' mechanism of action and resistance at single cell level.