The interest in ancestry informative markers as an investigative tool to identify the biogeographical ancestry of potential suspects is increasing. For this purpose, many different panels have been designed to distinguish individuals from major geographical regions. The EUROFORGEN North Africa and Middle East ancestry panel is a second-tier panel designed for differentiating Middle Eastern/North African populations from other population groups. An Ion AmpliSeq™ custom panel (Thermo Fisher Scientific) was designed to sequence populations of interest using the Ion S5™ (Thermo Fisher Scientific). Of the 111 SNPs selected in the first version of the panel, 107 SNPs were successfully incorporated in the AmpliSeq™ multiplex PCR. More than 600 individuals from 13 different countries in the Middle East, North Africa, and Europe were typed. The performance of the sequencing assay was assessed by calculating relative locus balances, heterozygote balances, and noise levels. Lastly, likelihood ratios were calculated using the GenoGeographer software.