Abstract: The AmpFℓSTR® Identifiler® Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA® card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay’s sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA® cards, and the assay’s specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA® substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler® Direct Kit for forensic standards and database samples genotyping.