U.S. Geological Survey 1 article published in JoVE Environment Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications Katy E. Klymus*1, Dannise V. Ruiz Ramos*1, Nathan L. Thompson*1, Catherine A. Richter*1 1Columbia Environmental Research Center, U.S. Geological Survey Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.