Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequence results. Here, we describe a reliable method for the preparation of mtDNA-enriched samples from eukaryotic cells ready for direct sequencing. This protocol utilizes a conventional miniprep kit, in conjunction with a paramagnetic bead-based purification step.