Mitochondrial disorders are among the most common inherited genetic disorders, with a combined prevalence of 1:5,000. These are genetically, biochemically, and clinically heterogeneous disorders affecting any organ or tissue in the body. A poor understanding of gene-to-phenotype relationships and pathophysiological mechanisms has resulted in sometimes years-long diagnostic odysseys and a lack of curative therapies. Consequently, outcomes are often poor with most patients dying in early childhood. The aim of this project is to improve patient outlooks by using novel tools to address both the diagnostic and therapeutic challenges associated with mitochondrial disease. The diagnostic aspect of the study involved the creation of four interactive diagnostic resources which can complement next generation sequencing (NGS) technologies to achieve more rapid diagnoses for patients. MitoEpilepsy Map, MitoCardio Map, MitoLiver Map, and MitoMedicine Map were created to aid in the diagnosis of mitochondrial epilepsy, cardiomyopathy, liver disease, and the entirety of mitochondrial disease, respectively. These, maps were accurate in identifying candidate genes from clinical vignettes of genetically confirmed cases of mitochondrial disease in 69-100% of cases. These maps will be valuable resources for interpreting NGS results, hopefully facilitating quicker and more accurate genetic diagnoses for affected patients. The therapeutic aspect of the project aimed to develop a new treatment strategy for mitochondrial disease caused by nonsense mutations. Translational read-through therapy involves pharmacological incorporation of a near-cognate amino acid in place of a premature stop codon during translation. A systematic in vitro proof-of-principle study was performed in patient fibroblasts harbouring bi-allelic nonsense mutations in ten different mitochondrial disease genes. In five patient cell cultures, translational read-through therapy was able to restore transcript, protein, and mitochondrial function, thus demonstrating in vitro efficacy and paving the way for future clinical development. Together, these approaches help improve outcomes for patients suffering from mitochondrial disease.