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The prevalence of the inclusion or substitution of porcine and its derivatives in foods by imprudent food manufacturers accentuated the need for a sensitive and specific method to detect porcine DNA. This work aimed to develop and validate an EvaGreen dye-based quantitative real-time (qPCR) assay for porcine DNA analysis. The primer set used in this work targets a 95-bp fragment of the porcine cytochrome b gene. Application of the assay to dilutions of porcine genomic DNA showed that the assay is sensitive down to 10 pg/μL of porcine DNA and the detection limit, under binary admixture conditions, was 0.001%. A correlation coefficient (R2) of 0.998 and PCR efficiency of 101.6% over the dynamic range from 10 to 10000 pg/μL indicated that the assay has high linearity and efficiency. The method’s specificity towards porcine was revealed by the amplification profile, which only displayed an amplification signal for porcine DNA. These findings suggest that the developed assay is sensitive, specific, and rapid. In conclusion, the assay can be used as an alternative method to detect and quantify porcine DNA in raw and processed food products.