ABSTRACT: Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) is a powerful technique for molecular analysis of surfaces; however, its application of single cell studies has not been previously published. In the current work, a commercial DESI setup (DESI XS) coupled to a mass spectrometer was used to analyze cultured mammalian cells attached onto glass coverslips. A series of experiments have been performed to obtain optimized experimental conditions, and MS images of metabolites with the single-cell level resolution were obtained using 10 µm x 10 µm pixel size. This established method can be readily adopted to extend the power of commercial DESI-MSI techniques in metabolomics studies requiring cellular resolution.