Nitric oxide (NO) is a small molecule that plays important roles in biological systems and human diseases. The abundance of intracellular NO is tightly related to numerous biological processes. Due to cell heterogeneity, the intracellular NO amounts significantly vary from cell to cell, and therefore, any meaningful studies need to be conducted at the single-cell level. However, measuring NO in single cells is very challenging, primarily due to the extremely small size of single cells and reactive nature of NO. In the current studies, the quantitative reaction between NO and amlodipine, a compound containing the Hantzsch ester group, was performed in live cells. The product dehydro amlodipine was then detected by the Single-probe single-cell mass spectrometry technique to quantify NO in single cells. The experimental results indicated heterogeneous distributions of intracellular NO amounts in single cells with the existence of subpopulations.