A laboratory testing algorithm was evaluated to confirm West Nile virus (WNV) infection in human serum following its introduction in Puerto Rico in 2007. This testing algorithm used two standard diagnostic assays, the IgM antibody capture enzyme linked immunosorbent assay (MAC ELISA) and real time reverse transcriptase polymerase chain reaction (RT-PCR), along with two non-conventional assays, the non-structural protein 1 (NS1) ELISA and plaque reduction neutralization test at 90% (PRNT(90)) with IgG depletion for dengue virus (DENV) and WNV. A total of 2321 serum samples from suspected WNV human cases were submitted for testing. Approximately one third (867, 37%) were cross reactive for DENV and WNV by MAC-ELISA and had negative RT-PCR results for both viruses. Of a subset of 43 samples tested, 31 (72%) of these cases were identified as dengue in the PRNT(90) IgG depletion assay and 8 (19%) were positive in the dengue NS1 antigen ELISA. These two assays combined differentiated 36 (84%) of the samples that could not be diagnosed using the standard diagnostic testing methods.