Neurotoxic shellfish poisoning caused by the ingestion of shellfish contaminated with brevetoxins threatens human health and economies reliant on the Gulf of Mexico. In the United States, the only federally approved method to test shellfish for brevetoxins is a mouse bioassay. Although effective in protecting public health, this assay has considerable shortcomings, including low throughput, lack of sensitivity and specificity, and reliance on live animal testing. Here, a commercially available enzyme-linked immunosorbent assay (ELISA) (MARBIONC) was validated, and results of field samples tested using ELISA and the mouse bioassay were compared. In this validation of the ELISA method for eastern oysters Crassostrea virginica and clams (Mercenaria spp. and sunray venus Macrocallista nimbosa), ELISA demonstrated strong recoveries, specificity, and ruggedness. Quantitative results were obtained for more than 90% of the 526 field samples tested by ELISA compared with only 26% of the samples tested by mouse bioassay. When quantitative data were available for both assays, significant correlations across methods were found. Based on this work, in 2017, the Interstate Shellfish Sanitation Conference approved the limited use of the MARBIONC ELISA in neurotoxic shellfish poisoning management.