Traditional Salmonella detection methods, including ISO 6579-1:2017 and ISO/TR 6579-3:2014, rely on enrichment and culture-based workflows that require 3–14 days to generate results. These conventional methods may not detect viable but non-culturable (VBNC) cells. Commercial PCR kits, while faster, often target highly variable virulence genes, leading to potential false negatives when applied to genetically diverse regional strains. This study presents a custom TaqMan real-time PCR assay specifically designed for Salmonella strains isolated across Indonesia. Primers and probes were developed to target genetically stable regions: the AHS-like conserved domain and iclR gene for Salmonella spp., and a Helix-Turn-Helix domain-containing protein for S. Enteritidis. A rapid DNA extraction protocol using lysis buffer and PBS wash was employed, eliminating the need for column purification and reducing sample preparation time. The assay was evaluated using 20 local Salmonella strains obtained from various food matrices, including egg, meat, dairy, fish, and processed products. While five strains were undetectable by commercial PCR kits, the custom assay accurately detected all 20. It demonstrated a limit of detection of 0.0001 ng/μL and produced results in less than 24 hours. The assay enables subspecies-level discrimination through primer design and includes internal controls for quality assurance. By targeting conserved genomic regions and avoiding culture dependency, this method enables the detection of VBNC strains and offers enhanced sensitivity, speed, and inclusivity. It represents a practical molecular tool for food safety surveillance, regulatory screening, and trade assurance, particularly in regions with unique pathogen profiles.