AMP-activated protein kinase (AMPK) is an evolutionally conserved key sensor of cellular energy status. Under conditions of cellular energy stress, AMPK is activated and will switch off anabolic processes that consume ATP, while activating catabolic processes that will generate ATP in order to restore energy balance. AMPK has been proposed as a drug target to treat metabolic disorders, and selective AMPK activators are being developed with the prospect of therapeutic application. In light of this, it is important to understand the molecular and physiological effects of AMPK activation in different cells/tissues. The aim of this thesis is to further our understanding of AMPK downstream actions through identification and characterization of novel AMPK substrates. In the studies encompassed in this thesis, we have investigated the effect of a dual treatment with two small molecule drugs on cellular AMPK activity and downstream action, and we have employed this pharmacological dual activation strategy to identify novel substrates of AMPK using two affinity proteomics-based approaches. We found that co-treatment of primary hepatocytes with two small molecule AMPK activators targeting distinct allosteric sites was able to robustly activate cellular AMPK. This treatment with submaximal dose of AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and low dose (1 ΌM) of A769662 produced a synergistic effect on AMPKα Thr172 phosphorylation and associated catalytic activity. Furthermore, the activation of AMPK activity by the co-treatment resulted in a more pronounced inhibition of lipogenesis in primary hepatocytes compared to single compound treatment. We further established two methodologies to identify AMPK targets based on: (1) immunoprecipitation with an antibody recognizing the optimal AMPK phosphorylation motif, and (2) ectopical expression of genetically engineered AMPK allowing specific labelling of direct phosphorylation targets. Using these two approaches, we identified putative AMPK substrates involved in a variety of cellular processes in mouse primary hepatocytes, such as mitochondrial dynamics and glycogen metabolism. We further identified Ser129 and Ser146 on mitochondrial fission factor (MFF), Ser902 on GTPase-activating protein and VPS9 domain-containing protein 1 (Gapex-5) and Ser175 on starch-binding domain-containing protein 1 (STBD1) as AMPK target phosphorylation sites, and further characterized them with respect to their AMPK-dependent regulation by small molecule activators, establishing them as genuine novel AMPK substrates. This work provides novel insights into the molecular mechanisms of AMPK downstream action in hepatocytes, and subsequent studies are starting to elucidate the physiological role of these regulatory events, although further work is still required. Additionally, the methodologies we and others have developed will allow further investigation of AMPK downstream effectors in a cell- and tissue-dependent manner. This will advance our understanding of the effects of AMPK activation by physiological and pathological stimuli, as well as in the context of pharmacological activation of AMPK for the treatment of metabolic disorders.