Three mitochondrial uncoupling proteins (UCP1, 2, 3) have been described. The proton transport activity of UCP1 triggers mitochondrial uncoupling and thermogenesis but the roles of UCP2 and UCP3 remain debated. Accordingly, compounds able to finely control the proton permeability of the mitochondrial inner membrane where and when needed may have enormous practical consequences. Using purified hamster brown adipose tissue UCP1 reconstituted in liposomes, we describe herein a robust assay allowing the measurement of this artificial membrane conductance to protons in a format compatible with high-throughput screening. The assay was initially developed with a known chemical protonophore in an aproteic system. Then, using the proteolipid reconstituted UCP1 preparation, we assessed the assay with known modulators of UCP1, particularly retinoic acid and guanosine 5'-triphosphate. The system was developed for a 96-well plate format. We then exemplified its use by generating primary data on a set of compounds screened in this system. These primary data will open new routes for the search of candidate compounds that will help biochemical studies on UCPs.