In vitro phosphorylation assays were conducted to identify phosphorylation sites on Dot1p catalysed by Hog1p. As several other kinases were co-purified with Hog1p, dead kinase control assays (Hog1p-K52R and Hog1p-D144A) were performed to verify the identified Dot1p phospho-sites. This entry contains the data pertaining to the in vitro Hog1p dead kinase phosphorylation assays, which is in supplement to existing PRIDE entry PXD036747.