Protein phosphorylation plays key roles in the regulation of normal and cancer cells. It is a highly dynamic process. Protein kinases are the targets of several new cancer drugs and drug candidates. However, some of the main issues related to new drugs are how they function and the selection of those patients that will likely respond best to a particular treatment regime. There is an urgent need to understand and monitor kinase signalling pathways. Phosphoproteomics requires the enrichment of phosphorylated proteins or peptides from tissue or bodily fluids, and the application of technologies such as mass spectrometry (MS) to the identification and quantification of protein phosphorylation sites. As the field develops it will provide pharmacodynamic readouts of disease states and cellular drug responses in tumour samples. There have been a number of recent advances, but there are still technical hurdles and bioinformatics challenges that need to be addressed.