Salmonella Typhi causes typhoid fever and requires prompt and precise diagnosis to prevent the disease from spreading. However, traditional methods for diagnosing the disease are time-consuming. The objective of this research is to assess the speed and reliability of Salmonella Typhi detection through multiplex PCR (mPCR) assay. Initially, Vitek2 system was used to identify 43 Salmonella Typhi isolates from fecal samples. These isolates were then subjected to PCR and mPCR, targeting genes of 16S rRNA, ropE, hil (hilA, hilC, hilD), and sip (sipA, sipB, sipC, sipD). The assay was also tested on seven non-Salmonella gram-negative bacteria known to cause diarrhea to verify the assay's specificity. Subsequently, mPCR was directly conducted on total gDNA extracted from patients' fecal samples to verify its effectiveness. The mPCR technique accurately amplified the target genes to the expected size and effectively distinguished between Salmonella and non-Salmonella isolates. The mPCR assay proved to be a reliable and rapid method for detecting Salmonella, significantly reducing the time required for diagnosis compared to traditional methods, allowing for timely and appropriate treatment of patients.