Forensic science takes advantage of population variability in autosomal Short Tandem Repeat (STR) lengths to establish human identification. The most common method for DNA profiling by STR is based on PCR, where the highly polymorphic STR regions are amplified and analysed using Capillary Electrophoresis (CE) or Massively Parallel Sequencing (MPS). MPS determines not only the repeat length, but also the repeat structure and variations in the flanking regions, making this method superior in discriminatory power compared to CE. Reverse Complement PCR (RC-PCR) is a novel, more sophisticated PCR based MPS library preparation method combining indexing and PCR amplification in a single closed-tube reaction. In this document we describe the complete developmental validation of the IDseek® OmniSTR™ kit, an RC-PCR based MPS library preparation kit. The developed IDseek® OmniSTR™ kit contains 28 autosomal STR targets, one Y-chromosomal STR and the Amelogenin gene covering all relevant STR core loci from the USA, EU, UK and Interpol.