Posttranslational modification of proteins is a key regulatory mechanism in a variety of cellular processes. This chapter outlines the concepts and methods used to investigate protein phosphorylation and its physiological relevance during plant virus infection. Rather than providing an exhaustive review of the experimental protocols for protein phosphorylation analysis, we focus on methods that can be used to study phosphorylation of viral proteins. We address the following points: how to determine that a viral protein of interest is phosphorylated; how to map the phosphorylation sites; how to identify the protein kinase(s) involved. Finally, we describe a number of useful strategies to evaluate the biological significance of phosphorylation.