Mitochondrial carriers are membrane proteins that transport keto acids, amino acids, fatty acids, nucleotides, vitamins and inorganic ions across the inner membrane of mitochondria.1 Here we discuss the use of thermostability shift assays to probe the transport and regulatory mechanism of mitochondrial carriers. In these assays, protein unfolding is monitored with the fluorescent dye that emits a fluorescent signal upon reaction with cysteine residues.2 A controlled temperature ramp is used to denature the population of membrane proteins, exposing hidden cysteine residues to generate a melting curve.2,3 Here we show that they can be used to study the nature of the interactions of lipids, detergents, substrates and inhibitors with mitochondrial carriers and uncoupling proteins.3,4 In combination with mutagenesis they can also be used to show state-dependent amino acid interactions as part of a transport cycle5 and protein domain interactions as part of a regulatory mechanism.1 Kunji ERS. Structural and Mechanistic Aspects of Mitochondrial Transport Proteins. In: Ferguson S, ed. Comprehensive Biophysics: Elsevier; 2012:174-205.2 Alexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC. Microscale fluorescent thermal stability assay for membrane proteins. Structure 2008;16:351-9.3 Crichton PG, Lee Y, Ruprecht JJ, et al. Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers. J Biol Chem 2015;290:8206-17.4 Lee Y, Willers C, Kunji ER, Crichton PG. Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin. Proc Natl Acad Sci U S A 2015;112:6973-8.5 King MS, Kerr M, Crichton PG, Springett R, Kunji ER. Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier. Biochim Biophys Acta 2016;1857:14-22.