Food fraud has become a widespread problem. In January 2013, the Food Safety Authority of Ireland uncovered a food fraud in which horse meat was being passed off as beef. In February 2013 and March 2014, the European Commission adopted a recommendation for a coordinated control plan in order to evaluate the prevalence of fraudulent practices. In this chapter, we describe a real-time polymerase chain reaction (PCR) method for the specific detection of horse (Equus caballus) and the development of a semi-quantitative PCR method, which was recommended for the two coordinated control plans. The developed PCR assay amplifies an 87 bp mitochondrial DNA fragment. The specificity, sensitivity and robustness of the PCR assay were successfully tested. As contamination between meat species is an unavoidable fact in the food industry, this semi-quantitative method was developed around a tolerance level set by the European Commission at 1% in mass fraction. The semi-quantitative method was intended to serve as a harmonized procedure that could easily be applied in the European Union, providing similar conclusions in different laboratories. While not every extraction method is suitable for this protocol, ways of checking the feasibility of the method have been provided.