Adulteration of dairy products, mainly through the substitution of high-quality milk for lower-quality milk, results in the production of low-value products, raising health, social, and economic concerns. As such, the development of methods to ensure dairy products' safety and quality is of great concern for governments and consumers. Although several methods have been developed for species differentiation in dairy products, their application and the establishment of reliable molecular markers for authentication purposes still need to be improved. In this chapter, we describe a low-cost, sensitive, fast, and reliable PCR-based method for mitochondrial D-loop DNA amplification for efficient detection of cattle milk in binary mixtures with sheep milk, thereby allowing the authentication of processed dairy products.