Factor V Leiden (FV-Leiden) and prothrombin gene mutations (FII G20210A) are well-established independent risk factors for several pregnancy complications and may be associated with an increased risk of recurrent miscarriage. Molecular diagnosis testing for both mutations is widespread and need to be standardized. We have optimized a duplex polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) for the simultaneous detection of both mutations in a single-tube single-enzyme digestion reaction for 35 women with recurrent miscarriage. This assay is a convenient method that should be applied in routine settings for detecting thrombophilia in women who are suffering from recurrent miscarriage.