Tick and tick‐borne diseases have become widely distributed throughout the United States. As a result, the interest associated with tick allocation and the potential threat they may pose has increased. Efforts have expanded to understand biotic and abiotic factors which may influence tick/pathogen distribution. Thus, we have developed a procedure which allows the simultaneous isolation of both tick and bacterial DNA. Downstream applications are diverse; however, we describe the use of multiplex PCR to confirm the presence of spirochete DNA from tick samples. We suspect that this procedure is not limited to tick‐bacteria systems and may be applied to a variety of arthropod‐related endeavors. Curr. Protoc. Microbiol. 19:1E.2.1‐1E.2.11. © 2010 by John Wiley & Sons, Inc.