Abstract: Little is known about the structure and physicochemical properties of the Infectious Laryngotracheitis Virus (ILTV) genome until recently a few years. To date, there is no protocol that is suitable for preparation of pure cell-associated herpesvirus DNA isolates from infected tissues or from tissue culture. The products of traditional methods are often found to be contaminated with host cellular DNA, especially for preparations of the large ILTV DNA genome. In addition, there is a need to develop methods for the isolation of highly purified viral DNA from cell culture or tissue samples to be used for high-throughput nucleotide sequencing. In this study, the isolation of ILTV from Chorioallantoic Membranes (CAM) was chosen as the model to test a method for purification of cell-associated herpesvirus DNA. The protocol was a combination of Triton X-100 lysis, nuclease treatment, nuclease denaturation, phenol-chloroform extraction and subsequent selective removal of small DNA fragments. The results showed that the cell background contamination was reduced significantly to a level that was suitable for 454 pyrosequencing and downstream applications such as cloning of viral DNA fragments or transfection of genomic viral DNA. This novel protocol, therefore has several advantages compared with existing methods.