AMP-activated protein kinase (AMPK) is the downstream component of a kinase cascade that senses cellular energy status. AMPK modulates multiple metabolic pathways and is activated by a large variety of cellular stresses, such as hypoxia and muscle contraction, or by adipokines and anti-diabetic drugs. In addition to its role in maintaining energy homeostasis,AMPK function is now recognized to extend to non-metabolic processes for example the control of cell structure and cell polarity. Elucidating the functional components of these complex signalling networks is a challenging prospect. Most of the bona fide AMPK substrates have been found by zooming in on candidate target proteins and validating them by classical biochemical approaches. As a complementary approach, phosphoproteomics can be used to identify new AMPK targets and identify their phosphorylation sites. As a proof of principle, this approach is being applied to full extracts from isolated hepatocytes treated with AICA riboside to activate AMPK. Tryptic peptides are first separated by the use of Hydrophilic-Interaction Liquid Chromatography (HILIC). Phosphopeptides are then selectively enriched on TiO₂ beads and analyzed by on-line reverse phase separation coupled to mass spectrometry. This will hopefully allow a comprehensive list of phosphorylation sites to be compiled and identify new AMPK targets. Phosphorylation of purified recombinant protein substrates by AMPK will be validated in vitro and phosphorylation of the targets in vivo will be confirmed after obtaining phosphorylation site-specific antibodies. Once validated, new areas of control by AMPK will be investigated.