MicroRNA (miRNA) genes have been shown to perform a crucial role in breast cancer metastasis. The epigenetic inactivation of such microRNA genes, as a result of aberrant DNA methylation, is frequently found in human tumours including those of the breast, and this is an area of considerable research activity. Pyrosequencing™ is a new quantitative method for the assessment of DNA methylation, with single CpG site resolution. Pyrosequencing™ can easily be performed in a 96-well-plate format with a cost-effective medium-sized throughput. This chapter provides a general outline of DNA methylation analysis, a detailed protocol of the Pyrosequencing™ procedure, and guidelines for the design of new assays. The strengths and limitations of this approach are discussed throughout the chapter.