Mitochondrial DNA (mtDNA) analysis is centralized in the Department of Neuropathology, Beaumont Hospital. Services offered include analysis of common mtDNA point mutations, large scale mtDNA deletions/rearrangements, and sequencing of the nuclear gene POLG. The aims of this study were to audit the mtDNA diagnostic service over a 10-year period, to determine appropriate use of the service, and to improve efficient use of the service by devising a requisition form that includes diagnostic algorithms. Between July 2002 and October 2013, 716 samples were received for analysis. Overall, the number of confirmed mutations was low. Lack of diagnostic algorithms may result in expansive, unrefined requests, leading to costly investigations. We introduced a requisition form that extracts clinical, biochemical, and pathological data prior to analysis. With this information, diagnostic algorithms can be applied to select the most relevant mutations for initial analysis and also to increase the incidence of mutation detection.