Mitochondrial diseases such as Pearson syndrome, Kearns-Sayre syndrome, and Chronic Progressive External Ophthalmoplegia are caused by structural rearrangements of the mitochondrial DNA (mtDNA); typically 2-8 kb deletions. The current testing strategy in the Oxford Medical Genetics Laboratories (OMGL) for these diseases is long-range PCR and Southern blot analysis. The aim of this project is to investigate whether a Next Generation Sequencing (NGS) approach can be used to determine the exact breakpoints of mtDNA deletions in order to provide further characterisation of these rearrangements and a more detailed diagnostic report. Two NGS technologies have been used in this study, the Illumina MiSeq and the Oxford Nanopore Technologies (ONT) MinION. Long-range PCR was used to generate a 16.1 kb fragment covering the majority of the mitochondrial genome in a selection of deletion patients and normal controls. A manual read depth analysis method was carried out on the subsequent MiSeq and MinION sequencing data in order to estimate the breakpoint locations for the single deletion patients. These breakpoints were then confirmed by bespoke PCR and Sanger sequencing. The results presented here show that both the Illumina and ONT NGS strategies can be used to accurately determine the breakpoints of single mtDNA deletions. Further work is required to investigate the possible role of bioinformatics tools in the automation of this breakpoint analysis. As a result of this work, one of these NGS strategies will be integrated into the OMGL diagnostic service to replace the need for Southern blotting in the characterisation of mtDNA deletions.