Abstract: Rat meat is not halal for Muslims, so that the presence of rat meat in any food is a crucial issue. The aim of this study was to design specific primer from Mitochondrial Cty b Rattus argentiventer that can be used for determining rat meat contamination or rat meat adulteration in meatball formulation using, Real Time Polymerase Chain Reaction (RT-PCR). The specificity of primers was confirmed in fresh tissue from pigs, cows, chickens, goats, rabbits and white mice. The designed primers were then used to analyze rat meat DNA in meatball formulation made from rat meat and beef mixture at 1, 2, 3, 5, 10, 25, 50 and 100% incorporation of rat meat. The repeatability test was performed by measuring the amplification from fresh rat tissue and rat meat in meatball. Primers were also subjected to sensitivity test of 6 dilution series (50000, 5000, 500, 50, 5 and 0.5 pg μL–1) of rat tissue. From two primers designed, primers cytb 42 (forward: 5’-TAA CCA CTC CTT CAT CGA CCT T-3’; reverse: 5’-CCC CGT TGG CGT GTA AAT A-3’) were more specific to evaluate the presence of rat meat in fresh tissue and in meatball formulation at optimum annealing temperature of 61.4°C. The primers can be used for DNA identification by RT-PCR with sensitivity expressed by limit of detection of 5 pg or in meatball formulation at concentration of 1% rat meat.