Plant organ abscission is a highly organized biological process in response to developmental and environmental cues. The understanding of the regulation of abscission is of great importance for the improvement of crop productivity and quality. The abscission process can be quantitatively analyzed by investigating the abscission of the tomato flower pedicel of different genotypes at a specific period. Single-cell transcriptomics is a novel approach to dissect the characteristics of pedicel abscission zone cells. In this chapter, we provide a detailed description of an abscission assay of flower pedicel explants and the method of single-cell transcriptome sequencing, including sample preparation, data determination, and analysis.