Mitochondrial diseases are a highly heterogeneous group of metabolic disorders that can present a clinical diagnostic challenge. A lack of rapid, reliable and sensitive diagnostic indicators often results in patients enduring testing by numerous clinical specialties, with no guarantee of a diagnosis and a possibility of misdiagnosis. In recent years, advances in mitochondrial disease diagnosis have been made and are becoming increasingly important as more and more clinical trials for mitochondrial disease therapies emerge. This seminar will present an Australian experience of advancing diagnostic approaches at the largest tertiary referral centre for adult mitochondrial diseases in Australia.