Factor V Leiden and prothrombin (F2) c.20210G>A mutation detection are very important in order to define the increased relative risk for venous thromboembolism in selected patients. Use of DNA-based methods to detect both mutations has become widely available in clinical diagnostic laboratories, including fluorescence-based quantitative real-time PCR (qPCR). The latter is a rapid, simple, robust and reliable method to identify genotypes of interest. There are several chemistries used for qPCR; this article describes their principles and applicability for Factor V Leiden and prothrombin (F2) c.20210G>A mutation detection.