Mitochondrial uncouplers are small molecules that 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 uncouplers increase energy expenditure / metabolic rate and have potential to be developed as pharmacotherapies for the treatment of obesity and metabolic disease. 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. However, medical prescription of DNP had to be banned by the U.S. Food and Drug Administration in 1938 due to significant adverse side effects. Although the toxicity associated with DNP is largely thought to be driven by off-target activity at non-mitochondrial membranes, its narrow therapeutic window has led to the common misconception that all mitochondrial uncouplers are dangerous. In the 80 years since DNP was banned there has not been an FDA-approved mitochondrial uncoupler for weight loss. Recently, the growing prevalence of obesity and metabolic disease, as well as the identification of novel uncouplers with improved safety profiles has renewed interest in uncoupling as a weight loss strategy. Dozens of new uncouplers have been identified, but few behave similarly in vitro and in vivo. The observed differences in behaviour may in part be due to a lack of standardisation in the methods and experimental systems used to characterise these molecules. Herein we directly compared fifteen mitochondrial uncouplers side-by-side in a well-defined in vitro system and then assessed the therapeutic potential of the top five candidates in the db/db mouse model of severe obesity and insulin resistance. We found that no two mitochondrial uncouplers behaved the same, with each molecule having a unique phenotype in terms of in vitro bioactivity and in vivo effects on body composition, glucose control and liver steatosis.