STR multiplexes remain the cornerstone of genotyping forensic samples. The PowerPlex 16 HS System contains the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. The PowerPlex 16 HS System is an updated version of the PowerPlex 16 System; while the primers and dyes remain unchanged, it introduces an enhanced buffer system that includes hot-start Taq DNA polymerase and ensures robust performance. Due to the modification of the reaction mix, a multi-laboratory developmental validation study was completed to document performance capabilities and limitations for the revised assay. Data within this validation was generated by eight laboratories and served as the basis for the following conclusions: genotyping of single-source samples was consistent across a large range of template DNA concentrations with most laboratories obtaining complete profiles at 62.5pg. Mixture analyses showed that over 90% of minor alleles were detected at 1:9 ratios. Optimum amplification cycle number was ultimately dependent on the sensitivity of the detection instrument and could be adjusted to accommodate a range of DNA template concentrations. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, Taq DNA polymerase, and magnesium were shown to be optimal and able to withstand moderate variations without affecting multiplexed STR amplification. Finally, data from non-probative samples and concordance studies showed consistent results when comparing the PowerPlex 16 HS System with the PowerPlex 16 System as well as other commercially available systems.