Two assays (Liaison, Diasorin; IDEIA, Oxoid) for detection of Borrelia-specific antibodies were compared. A case-control design using patients with neuroborreliosis (n = 48), laboratory defined by a positive Borrelia-specific antibody index in the spinal fluid, was available and was intended to represent the serological response of disseminated early Lyme borreliosis in general. Serum samples were obtained from 216 Danish blood donors as controls. By comparing sensitivity and specificity using pre-specified cut-off values, significant differences were found. However, using receiver operating characteristic (ROC) curves to optimize and standardize test interpretation, it was shown that testing with both IDEIA IgG and IgM was comparable to testing with Liaison IgG alone by comparing the area under the curve of the diagnostically relevant 25 % partial ROC curve (P = 0.1). When using the Liaison OspC/VlsE IgM assay, the specificity was decreased without a gain in sensitivity. This study proposes standardizing of reporting by using a control population as the reference and choosing decision thresholds guided by the risk of false-positive results at 2 and 8 %. The sensitivities for IDEIA (IgG and IgM combined) were 85 and 95 % and for the Liaison (VlsE IgG) method were 67 and 96 %, respectively. Methods for test evaluation, test interpretation and statistical testing are presented and discussed. In conclusion, Liaison VlsE IgG alone and IDEIA IgG/IgM combined showed a high and comparable discriminatory ability to distinguish serum samples from patients with neuroborreliosis from blood donor controls. However, cut-off values should be adjusted for a proper comparison.