Despite continuous conservation efforts by national and international organizations, the populations of the three extant elephant species are still dramatically declining due to the illegal trade in ivory leading to the killing of elephants. A requirement to aid investigations and prosecutions is the accurate identification of the elephant species from which the ivory was removed. We report on the development of the first fully validated multiplex PCR-electrophoresis assay for ivory DNA analysis that can be used as a screening or confirmatory test. SNPs from the NADH dehydrogenase 5 and cytochrome b gene loci were identified and used in the development of the assay. The three extant elephant species could be identified based on three peaks/bands. Elephas maximus exhibited two distinct PCR fragments at approximate 129 and 381 bp; Loxodonta cyclotis showed two PCR fragments at 89 and 129 bp; and Loxodonta africana showed a single fragment of 129 bp. The assay correctly identified the elephant species using all 113 ivory and blood samples used in this report. We also report on the high sensitivity and specificity of the assay. All single-blinded samples were correctly classified, which demonstrated the assay's ability to be used for real casework. In addition, the assay could be used in conjunction with the technique of direct amplification. We propose that the test will benefit wildlife forensic laboratories and aid in the transition to the criminal justice system.