Short Tandem Repeat (STR) analysis is a widely used method for human identification in forensic and medical applications. However, the precision and efficiency of this method rely extremely on the quality and reliability of the allelic ladder used for data interpretation. The performance of the Global FilerTM PCR Amplification Kit has been tested using 40 samples from various populations and found that it outperformed commonly used ladders in terms of allele calling, resolution, and sensitivity. Using DNA samples from 40 individuals, including five males and thirty-five females from various ethnic groups, the performance of the novel ladder was validated. A success rate of 99.5% and an average allelic dropout rate of 0.2%, the new ladder was highly precise and efficient, according to the results. The allelic ladder in this study has the accuracy and efficiency of STR analysis in forensic and medical applications, and it could be a valuable tool for human identification in a wide range of settings. For the advancement of forensic research, the development and validation of a better allelic ladder for precise and fast human identification utilizing STR analysis are crucial. This study’s novel ladder showed considerable gains over current ladders and had the potential to improve accuracy.