DNA analysis using advanced molecular biological tools is universally used to unlock identification information contained in crime scene stains. These advances have allowed scientists to provide critical information to solve crimes and allow the criminal justice community to convict the guilty and justify the innocent. However, despite the widespread use of trace DNA, at present there are very few specific validated methods. This has led to controversy in the use of trace DNA [[1]Forensic Genetics Research Progress.]. DNA profiling by targeting STRs present in the human genome has proven to be an extremely discriminating method for human identification in forensic and paternity applications for decades. It has been widely used for the identification of individuals based on their DNA characteristics [[2]Ludeman M.J. Zhong C. Mulero J.J. Lagacé R.E. Hennessy L.K. Short M.L. et al.Developmental validation of GlobalFiler™ PCR amplification kit: a 6-dye multiplex assay designed for amplification of casework samples.]. However, STR typing is limited by the quality of human DNA obtained from forensic casework samples that can be influenced by environmental factors which may cause different degrees of degradation, that have a negative impact on the amplification process especially of STR systems with large amplicons [[3]Senge T. Madea B. Junge A. Rothschild M.A. Schneider P.M. STRs, mini STRs and SNPs – a comparative study for typing degraded DNA.]. In this study, an evaluation of a six-dye STR multiplex assay (VeriFiler™ Plus) was conducted with casework samples; this assay is composed of the following markers: 23 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, D10S1248, D1S1656, D12S391, D2S1338, D6S1043, Penta D, Penta E), 1 insertion/deletion polymorphic marker on the Y chromosome (Y indel) and Amelogenin (sex-determining marker) using a 3500 genetic analyzer for fragment detection that include the analysis of the following parameters and aspects: sensitivity, robustness, analytical threshold, stochastic threshold, heterozygous balance, precision and accuracy, repeatability and reproducibility, genotype concordance, DNA mixtures studies and stability study.