Chemical protonophores transport protons across membranes. Protonophores that act at the mitochondrial inner membrane are referred to as mitochondrial ‘uncouplers’ because they disrupt the proton cycle that couples nutrient oxidation to adenosine triphosphate (ATP) production. In the presence of mitochondrial uncouplers, more nutrients must be oxidised to produce a given amount of ATP. Thus, mitochondrial uncoupling increases metabolic rate and represents a strategy for anti-obesity drug development. Small molecule mitochondrial uncouplers have been shown to decrease ROS production by facilitating proton leak and consuming the proton motive force. This mitochondrial anti-oxidant phenotype offers a secondary mechanism through which uncouplers could be of utility in the treatment of metabolic diseases such as diabetes and non-alcoholic steatohepatitis in which oxidative stress and inflammation are a major driving force in aetiology and pathogenesis. Humans taking the mitochondrial uncoupler 2,4-dinitrophenol (DNP) experienced an 11% increase in metabolic rate for every 100 mg ingested and lost 1.5 kg/week. Despite these promising results, medical prescription of DNP was banned by the U.S. Food and Drug Administration (FDA) in 1938 due to adverse side effects caused by off-target protonophore activity at non-mitochondrial membranes, including the plasma membrane. In the 80 years since DNP was banned, there has not been an FDA approved mitochondrial uncoupler for weight loss or treatment of metabolic disease. Recently, the growing prevalence of obesity and the identification of novel uncouplers with improved safety profiles has renewed interest in uncoupling as a therapeutic strategy. Dozens of new uncouplers have been described in the past decade, but they have been characterised under variable conditions making it difficult to compare their efficacy and toxicity. Here we compared 15 mitochondrial uncouplers side-by-side in vitro and then screened leading candidates in a db/db mouse model to identify the molecules that may be best suited for future drug development.