The Ingenomics™ AutoProfiler system utilises a 6-dye, multiplex genotyping technology that is optimised for the simultaneous amplification of 20 Combined DNA Index System (CODIS) short tandem repeat (STR) markers, along with D6S1043, Penta D, Penta E, DYS391, SE33, and two Y-Indels (Rs2032678, Rs771783753), as well as the Amelogenin loci. This kit includes specialised internal quality control (IQC) markers, both small and large, to evaluate the quality parameters for each sample. Designed for a diverse range of applications—such as forensic DNA casework, identity establishment, database management, kinship testing, scientific research, and other analyses related to human genetic identification—this multiplex system is particularly effective for analysing low-copy number (LCN) DNA from severely compromised forensic samples, as it also evaluates two SNP markers. The multidimensional validation study aligned with the guidelines set forth by the Scientific Working Group on DNA Analysis Methods (SWGDAM) examined crucial parameters, including PCR conditions, reproducibility, analytical threshold calculation, sensitivity, species specificity, stability, mixture analysis, casework sample analysis, direct amplification, and concordance. The optimal DNA concentration range was between 60 and 500 pg/µL, with an analytical threshold established at 50 relative fluorescence units (RFUs). Overall, the findings indicate that the Ingenomics™ AutoProfiler kit is a reliable, robust, and suitable assay for human identification in casework DNA analysis and database construction.