As short tandem repeat markers remain the foundation of human identification throughout the world, new STR multiplexes require rigorous testing to ensure the assays are sufficiently robust and reliable for genotyping purposes. The PowerPlex 18D System was created for the direct amplification of buccal and blood samples from FTA storage cards and reliably accommodates other sample materials. The PowerPlex 18D System allows simultaneous amplification of the 13 CODIS loci and amelogenin along with 4 additional loci: Penta E, Penta D, D2S1338, and D19S433. To demonstrate suitability for human identification testing, the PowerPlex 18D System was tested for sensitivity, concordance, inhibitor tolerance, and performance with thermal cycling and reaction condition variation following SWWGDAM developmental validation guidelines. Given these results, PowerPlex 18D System can confidently be used for forensic and human identification testing. (Published Abstract)