Using a chemical genetic approach, we identified four novel physiological substrates of Hog1 kinase (Krs1, Tdh3, Hsp26, and Shm2). These substrates suggest plausible mechanisms for actin reorganization, cell cycle arrest and regulation of protein synthesis observed upon osmotic stress. We further show that the human homolog of Shm2 (SHMT1) is a novel physiological substrate of p38 MAP kinase in vitro and in vivo. Down-regulation of its enzymatic activity was observed following p38-mediated phosphorylation revealing a potential cancer-modulating property of p38 MAP kinase. This screen has uncovered several novel Hog1 substrates that provide new avenues for investigation into the mechanism of osmoadaptation by this kinase.