Sexual assault is a terrible crime that necessitates a thorough and qualified investigation to collect the perpetrator's biological material. Forensic investigation is required as soon as possible to gather all possible physical and biological evidence. The primary focus of such investigations is the detection of male-specific materials, particularly sperm in semen stains. Advances in DNA-based testing now allow for on-site analysis at crime scenes. In this study, we developed an assay using the loop-mediated isothermal amplification (LAMP) technique to detect male DNA. The assay underwent validation following the SWGDAM guidelines and was subsequently tested on 92 casework samples from sexual assault cases. To evaluate its performance, we compared the results of three different tests: acid phosphatase, microscopic examination, and the LAMP assay, against the Y-STR profiling results. Findings from this study demonstrate the high efficiency of the LAMP assay when compared to Y-STR profiling. These results highlight the potential of the LAMP technique as a valuable tool for male DNA detection in forensic casework. Further research and validation studies are necessary to fully explore its practical applications and enhance its utility in criminal investigations.