Updated publication reference for PubMed record(s): 28270554. Saccharomyces cerevisiae reacts to elevated external osmolarity by several cellular responses. Thereby, the main adaptive signaling activity relies on the high-osmolarity glycerol (HOG) pathway, whose core architecture is closely related to the mammalian p38 MAPK pathway. To identify novel target proteins of its MAP kinase Hog1, we applied an MS-based high throughput analysis measuring the impact of Hog1 activation as well as inhibition on the budding yeast phosphoproteome. In addition, we analyzed how a deletion of RCK2, a known effector protein kinase target of Hog1, modulates the normal stress induced phosphorylation pattern. Our results provide an overview on the diversity of cellular functions that are directly and indirectly affected by the activity of the pathway and allow a clear assessment of Hog1 independent events affected by osmotic stress. Analyzing the modulation of S/T-P motifs, we could extend the number of Hog1 putative direct targets that were subsequently validated by an in vivo interaction assay. It also appears that Rck2, a downstream kinase of Hog1, acts as a central hub for many Hog1-mediated secondary phosphorylation events.