Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. Modification of target protein properties by reversible phosphorylation events has been found to be one of the most prominent cellular control processes in all organisms. The phospho-status of a protein is dynamically controlled by protein kinases and counteracting phosphatases. Therefore, monitoring of kinase and phosphatase activities, identification of specific phosphorylation sites, and assessment of their functional significance are of crucial importance to understand development and homeostasis. Recent advances in the area of molecular biology and biochemistry, for instance, mass spectrometry-based phosphoproteomics or fluorescence spectroscopical methods, open new possibilities to reach an unprecidented depth and a proteome-wide understanding of phosphorylation processes in plants and other species. In addition, the growing number of model species allows now deepening evolutionary insights into signal transduction cascades and the use of kinase/phosphatase systems. Thus, this is the age where we move from an understanding of the structure and function of individual protein modules to insights how these proteins are organized into pathways and networks. In this introductory chapter, we briefly review general definitions, methodology, and current concepts of the molecular mechanisms of protein kinase function as a foundation for this methods book. We briefly review biochemistry and structural biology of kinases and provide selected examples for the role of kinases in biological systems.