Molecular techniques for marine pest surveillance offer cost and time savings over traditional techniques, but require validation. qPCR assays are designed to provide specific detection using sequence data from the target and close relatives. Initial validation after design involves laboratory investigation of assay performance and demonstration of assay specificity against DNA of non-target species. Field validation is then carried out to ensure a lack of false positives in environmental samples and to demonstrate that each assay can detect the target pest in plankton from areas where they occur. Assays for pests that are exotic to Australia, or which occur only in restricted localities, however, cannot be field validated with natural samples. These species are not known to be present and so not expected to be detected, but for non-detections to be considered evidence of continued species absence, it is important to demonstrate that the assays would detect the target were it present. We developed a method for validating assays for exotic pests by spiking plankton samples with freeze-dried pest tissue in environmentally relevant amounts. Using this method, we validated the performance of qPCR assays for 12 species: 8 regarded as exotic to Australia, and 4 with restricted distributions.