Many genetic variants with association to normal pigmentary variation have been identified. Several assays for prediction of pigmentary traits have been developed using various types of genotyping methods, including Single Base Extension (detected by CE or MALDI-TOF MS) and real-time PCR. To circumvent the limited multiplexing capacity of these methods, we designed the Human Pigmentation (HuPi) AmpliSeq™ custom panel for the Ion S5™ System. The panel was designed as one large multiplex PCR using the Ion AmpliSeq™ Designer v7.0.6 (Thermo Fisher Scientific). The PCR amplifies 183 genetic variants with association to normal pigmentary variation of the eyes, skin, and hair in 46 genes including OCA2, MC1R, SLC24A4, SLC45A2, TYR, ASIP, HERC2, and TYRP1. The HuPi panel consists of 161 amplicons with an average amplicon size of 118 bp (70–137 bp), which makes the panel applicable for forensic casework. For the initial evaluation of the panel, we successfully typed 68 individuals. Fifteen loci were excluded due to low or no coverage (<30x). The heterozygote balances were around 1 for most loci, and the median level of noise <1%. Profiles with 168 loci were obtained from only 100 pg input DNA. In conclusion, the HuPi panel is a promising new assay for typing of pigmentary markers.