Rat meat (RM) can be obtained freely from farmers and can be used as a potential adulterant in meat-based food products. The presence of RM in any food products is prohibited by the Muslim community and is considered non-halal meat. Therefore, an analytical method capable of analysing RM specifically is very urgent. In this study, a real -time polymerase chain reaction using a species-specific primer targeting NADH dehydrogenase subunit 6 (ND6) is applied to analyze RM. DNA extraction was carried out using FavorPrepTM Tissue Genomic DNA Extraction Mini Kit, and the extracted DNA was subjected to purity index using NANO-Quant SPARK TECAN. The annealing temperature (Ta) used for PCR analysis was optimized to get the best Ta capable of providing the optimum amplification reaction. Furthermore, some performance characteristics were evaluated for Real-Time PCR, including sensitivity, efficiency and repeatability. The results exhibited that the designed primer is specific to rat DNA extracted toward other DNAs from meats typically used in food products. For quantitative evaluation, the limit of detection found was 0.39 ng with an efficiency of amplification (E) of 94.4% with a coefficient of determination (R2 ) of 0.986 for the relationship between log concentrations of DNA and cycle threshold (Ct) values. The repeatability assay was acceptable with an RSD value ≤ 25%. The developed method was reliable, provided high sensitivity for the analysis of RM. Therefore, this method is suitable to be used as a standard method for halal authentication analysis.