Prenatal diagnosis has become a standard part of obstetrics care. Genetic diagnoses are established prenatally through the sampling of fetal genetic material by invasive methods such as amniocentesis or chorionic villus sampling, which are associated with a risk of fetal loss. Hence, the recent discovery of the presence of fetal DNA in maternal plasma has led to exciting possibilities of noninvasive prenatal diagnosis. Numerous applications have since been reported for the analysis of circulating fetal DNA. The accuracy of fetal DNA assessment from maternal plasma is dependent on the appropriate preanalytical handling of maternal blood samples, an efficient fetal DNA extraction protocol, and a sensitive and specific detection method. The protocol that has been applied regularly in the laboratory of the authors for the reliable detection and quantification of circulating fetal DNA is presented in this chapter.