A rapid and efficient procedure for the purification of myxoma virus DNA from infected cell cultures is described. The traditional method used for recovery of myxoma virus DNA involves multiple freeze-thawing cycles to disrupt cells and release virions followed by ultracentrifugation to concentrate virions for DNA extraction. Freeze-thaw cycles are time consuming and reduce viral titers, while ultracentrifugation steps limit the number of samples that can be processed at one time, reducing efficiency. In this report an optimized method circumventing the time-consuming techniques and replacing them with rapid, efficient steps adequate for the processing of larger numbers of samples is described. The traditional method was compared with the optimized protocol, which was found to be more efficient in terms of time required to complete the process and in the quantities of DNA purified.