Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed due to the length of time required to obtain results. Polymerase chain reaction (PCR), more specifically the real-time detection of the amplified PCR product, is a methodology that can provide a diagnosis in a timelier manner compared to culture and serology. There are a limited number of real-time PCR (qPCR) assays for detecting Leptospira and not all of these assays are able to distinguish pathogenic from nonpathogenic species. In addition, there are a variety of probe technologies and qPCR instruments that are utilized with these assays. This chapter presents a qPCR assay that targets lipL32 , a gene which is present only in pathogenic Leptospira spp. This assay utilizes a TaqMan probe and instructions for use on either the Lightcycler 1.2 (Roche Diagnostics, Indianapolis, IN) or the ABI 7500 (Applied Biosystems, Foster City, CA) are provided.