A pig-specific real-time PCR assay based on the mitochondrial ND5 gene was developed to detect porcine material in food and other products. To optimize the performance of assay, seven commercial TaqMan master mixes and two real-time PCR platforms (Applied Biosystems StepOnePlus and Bio-rad CFX Connect) were used to evaluate the limit of detection (LOD) as well as the PCR efficiency and specificity. The LODs and PCR efficiencies for the seven master mixes on two platforms were 0.5-5 pg/reaction and 84.96%-108.80%, respectively. Additionally, non-specific amplifications of DNA from other animal samples (human, dog, cow, and chicken) were observed for four master mixes. These results imply that the sensitivity and specificity of a real-time PCR assay may vary depending on master mix and platform used. The best combination of master mix and real-time PCR platform can accurately detect 0.5 pg porcine DNA, with a PCR efficiency of 100.49%.