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Meat adulteration and admixing are prevalent malpractices observed in processed and raw meat samples, where the consumption of adulterated meat has been associated with food allergies, financial losses, and consumer distrust. Meat authentication is pivotal to address these concerns. The meat authenticity can be determined through genetic, protein, and immunological markers and advanced detection methods. However, these methods often target a single species and lack the specificity to distinguish closely related species. Here, in the present study, we have developed a multiplex detection method based on the species-specific primers and probes, that can target four meat species in one reaction. The developed method amplifies the mitochondrial genomic regions of chicken, pork, sheep and goat using TaqMan multiplex probe-based RT-qPCR assay. Unique pairs of species-specific primers and probes that target specific mitochondrial DNA (mtDNA) regions of each species were designed and screened for specificity and sensitivity. The detection limit for species identification using the designed primers in real-time qPCR assays was 0.1 picogram per microliter (pg/μL) DNA detected in singleplex reaction and facilitates the simultaneous detection of closely related species, such as goat and sheep. Further, DNA-based probes were utilized in a multiplex real-time qPCR assay to identify chicken, pork, sheep and goat DNA in a single tube reaction. The multiplex assay was validated for raw and processed meat products, demonstrating its applications in ensuring the quality of meat products and safeguarding consumer interests.