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. Guard cells are specialized epidermal cells that play an important role in gaseous exchange in and out of plant leaves by regulating the opening and closing of the stomata, and provide a model genetic system for analyzing cell fate, signaling and function. Several proteomics analyses of guard cells are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich for guard cell and mesophyll cell protoplasts and acquire in-depth proteomics in these two major cell types in Arabidopsis leaves. We identified ~3,000 proteins not previously found in the guard cell proteome and more than 500 proteins that may be specific to guard cells. The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf-like kinase15(RAF15) and Snf1-related kinase2.6(SnRK2.6)/OST1(open stomata 1) mediate abscisic acid (ABA)-induced stomatal closure. RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the dephosphorylated inactive form of SnRK2.6/OST1. ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in guard cells and represents a resource potentially providing many more insights into the molecular basis of guard cell and mesophyll cell development, structure, and function.