Vibrio parahaemolyticus (Vp) and V. vulnificus (Vv) are the leading causes of shellfish consumption-associated infections and mortality in the United States. The current National Shellfish Sanitation Program (NSSP)-approved DNA probe/colony hybridization method for isolate confirmation cannot be conducted as the manufacturer of the alkaline phosphatase (AP) probe has discontinued production. The AP probe is used in the NSSP for post-harvest process (PHP) validation/verification sample testing. It is important to find a cost-effective replacement for the AP method so PHP testing can continue to provide confidence that these processes are reducing the Vibrio pathogens to “non-detectable” levels in shellfish. Loop-mediated isothermal amplification (LAMP) was identified as a possible replacement. LAMP occurs at a constant temperature and amplification can be seen by the naked eye so expensive thermal cyclers are not needed, and it can be highly specific with up to six sets of primers per target species. Published LAMP assays targeting toxR for Vp and vvhA for Vv were evaluated and optimized for enzyme system, amplification temperature, and DNA preparation method against a panel of 185 isolates. Optimal conditions were tested for specificity and sensitivity. When non-specific amplification was seen in LAMP, chromogenic and selective agars were evaluated as a prescreening step to prevent non-target species from being tested in LAMP assays. Prescreening with chromogenic and selective agars increased specificity. Upon further testing, the toxR assay detected 49/50 (98%) Vp isolates and the vvhA assay detected 48/50 (96%) Vv isolates. Isolates that were not detected did not look typical on chromogenic or selective agars and were not picked to LAMP for confirmation. The completed research demonstrates that the published LAMP assays tested would need to be redesigned for increased specificity so prescreening would not be necessary before moving forward.