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liquid chromatography tandem mass spectrometry This article has not been copyedited and formatted. The final version may differ from this version. Abstract Valproic acid (VPA) is a widely prescribed anti-convulsant for the treatment of epilepsy. Here we demonstrate that VPA is a novel activator of AMP-activated protein kinase (AMPK), a key regulator of cellular metabolism, using primary mouse and human hepatocytes. Incubation of primary mouse hepatocytes with VPA resulted in increased levels of phosphorylated AMPK and acetyl-CoA carboxylase (ACC). This finding was recapitulated using primary human hepatocytes. Pre-treatment of mouse hepatocytes with small molecule inhibitor of AMPK, Compound C, abrogated the phosphorylation of ACC following treatment with VPA. The cytochrome P450 inhibitor 1-aminobenzotriazole blocked the VPA-stimulated phosphorylation of AMPK, suggesting a requirement for biotransformation of VPA. In line with this, treatment of hepatocytes with metabolites of VPA resulted in increased phosphorylation of AMPK/ACC as compared to VPA. Treatment of ob/ob mice with VPA for 14 days resulted in decreased liver masses, hepatic fat accumulation, and serum glucose. These results paralleled those observed in mice treated with metformin. In addition, a targeted mass spectrometry-based metabolomics assay revealed several small molecules that were differentially abundant in the serum of ob/ob mice treated with VPA as compared to vehicle-treated mice. These studies are the first to establish VPA and its metabolites as in vitro activators of AMPK. This article has not been copyedited and formatted. The final version may differ from this version.