Diagnostic testing for Lyme disease in the clinical setting primarily relies on assessment of serologic responses to infection, with the exception of the early localized phase of disease, in which the diagnosis must be made clinically, due to the recognized insensitivity of serologic testing at this phase of disease. For the diagnosis of early disseminated and late disease, the Centers for Disease Control and Prevention (CDC) recommends a two-tiered approach to testing consisting of initial IgM and IgG quantitative enzyme-linked immunosorbent assay (ELISA), followed by confirmation of all indeterminate or positive ELISA tests with separate IgG and IgM Western blots. This critical analysis addresses the sensitivity, specificity, and predictive value of serologic testing for Lyme disease in early localized, early disseminated, and late disease. Other testing modalities currently under evaluation are also discussed, including IgG vlsE C6 peptide ELISA, other two-tiered testing strategies, rapid diagnostics, and PCR. An understanding of the strengths and limitations of currently available testing for Lyme disease is critical for appropriate diagnosis.