Three types of meatballs were prepared following Rahman et al. (2014) and Rohman et al. (2011). First category beef meatballs were prepared by spiking 1% of each target species separately and the second type included all target meat species to make a total of 5% adulterated beef meatball and the third category was made including only with five target meats. Each meatball was made using 90% emulsified ground meat/meats (target meat species and beef) and 10% of starch mixed with relevant amount of salts and spices such as pepper, garlic, onion etc. (Table 2). Prepared meatballs were subjected to autoclave at 121 °C under 45-psi pressure for 2.5 h to simulate extensive cooking and boiling effects. All samples were prepared in triplicate on three different days by three independent analysts and were stored at −20 °C prior to DNA extraction.