In this study, we describe the developmental validation assay performed on a novel designed STR multiplex system, AGCU 21+1 STR kit. This kit contains a sex-determining locus amelogenin and 21 noncombined DNA index system STR loci, that are, D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D1S1627, D3S4529, D2S441, D6S1017, D4S2408, D19S433, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D9S1122, D2S1776, D10S1435, and D5S2500. The 21+1 kit was validated by a series of tests including optimized PCR conditions, sensitivity, precision and accuracy, stutter ratio, DNA mixture, inhibitors, and species specificity according to the revised validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Our results in this study show that the kit is a useful tool for forensic application.