Although methods are in place through the U.S. Fish and Wildlife (USFWS) program for ploidy testing of feral caught Grass Carp (Ctenopharyngodon idella) and black carp (Mylopharyngodon piceus), no guidelines exist for carp hauled across state lines. Using 1200 Grass Carp purchased by undercover Ohio law enforcement during 2015–2016, we developed a standardized protocol for discriminating ploidy by using two parameters, nuclear size and DNA content. Bead standards at 2 μm or 4 μm were used to establish nuclear size from Nile Tilapia (Oreochromis niloticus), known diploid (n = 20) and triploid (n = 20) Grass Carp blood, and cells derived from eyeballs of purchased field carp. The control for establishing DNA content was cryopreserved or fresh tilapia blood (2.40 pg). Time postmortem indicated nuclear size was similar over 4 days, but DNA quality from triploids was best at 24 h. Occasionally, only size or DNA content was measurable. Tilapia mean nuclear size (n = 501 samples) was 4.61 μm (SE 0.05) (R² = 0.94). Known diploid and triploid blood nuclear sizes, compared with tilapia size, were 3.62 μm (SE 0.13) (R² = 0.96) and 7.58 μm (SE 0.27) (R² = 0.96), respectively. Mean field carp eye nuclear size was 5.83 μm (SE 0.13). Mean DNA content of cells from field carp eyes (n = 698 fish) was 3.51 pg (SE 0.06). No diploid Grass Carp were detected in the USFWS certified triploid Grass Carp transports. This standard protocol reliably discriminates ploidy and can be used for enforcement of regulations that differ among state jurisdictions.