Standard 2-tier testing (STTT), incorporating a screening enzyme immunoassay (EIA) or an immunofluorescence assay (IFA) that reflexes to IgM and IgG immunoblots, has been the primary diagnostic test for Lyme disease since 1995. In 2019, the Food and Drug Administration approved a modified 2-tier test strategy using 2 EIAs: offering a faster, less expensive, and more sensitive assay compared with STTT. New technologies examine early immune responses to Borrelia burgdorferi have the potential to diagnose Lyme disease in the first weeks of infection when existing serologic testing is not recommended due to low sensitivity.