Although serologic testing remains the gold standard for laboratory diagnosis of Lyme disease, the antibody response may take several weeks to increase greater than the limit of detection. Because of this extended time frame, it is necessary to identify new diagnostic methods for earlier diagnosis and appropriate treatment of Lyme disease. Alternative diagnostic modalities, such as Borrelia culture or nucleic acid amplification testing, may be beneficial in specific clinical scenarios. In early phases of acute infection, before the development of an immune response, detection of Borrelia DNA from clinical specimens may help establish the diagnosis sooner than serologic methods.