The reversible phosphorylation of proteins controls virtually all aspects of cell function. However, in order to establish that the phosphorylation of a protein by a particular protein kinase is of physiological relevance, a series of criteria (proposed by Krebs & Beavo, 1979 ) should be satisfied. Surprisingly, amongst the thousands of protein kinase targets that have been reported in the literature, there are not so many for which there is good evidence for phosphorylation having functional consequences in vivo. Here we review the approaches that can be used to establish physiologically important protein phosphorylation according to the Krebs and Beavo criteria, taking as an example heart 6-phosphofruco-2-kinase phosphorylation-induced activation by insulin. We also point out the pitfalls of the various techniques that can be used to implicate the involvement of a particular protein kinase in a biological response. Lastly, we discuss the use of mass spectrometry techniques to search for new protein kinase targets, bearing in mind that each new target found would have to be validated by the criteria before being considered as a bona fide protein kinase substrate.