The PhosPhAt 4.0 database contains information on Arabidopsis phosphorylation sites identified by mass spectrometry in large-scale experiments from different research groups. So far PhosPhAt 4.0 has been one of the most significant large-scale data resources for plant phosphorylation studies. Functionalities of the web application, besides display of phosphorylation sites, include phosphorylation site prediction and kinase-target relationships retrieval. Here, we present an overview and user instructions for the PhosPhAt 4.0 database, with strong emphasis on recent renewals regarding protein annotation by SUBA4.0 and Mapman4, and additional phosphorylation site information imported from other databases, such as UniProt. Here, we provide a user guide for the retrieval of phosphorylation motifs from the kinase-target database and how to visualize these results. The improvements incorporated into the PhosPhAt 4.0 database have produced much more functionality and user flexibility for phosphoproteomic analysis.