Multicellular organisms such as plants contain different cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances understanding of organization and function at the organismal level. Here we used enzymatic isolation and flow cytometry-activated cell sorting (FACS) to enrich the guard cell and mesophyll cell protoplasts and acquire in-depth proteomics in these two major cell types in Arabidopsis leaves. We uncovered a guard cell-specific kinase cascade whereby RAF15 and OST1 mediate ABA-induced stomatal closure. This pipeline can be applied to various cell types in plant or non-plant systems to learn how cells function in highly organized multicellular organisms.