There is an increasing demand for genetic testing of patients with hypercoagulability. We have developed a multiplex-polymerase chain reaction (PCR) technique that incorporates the state-of-the art of genetic testing for thrombosis mutations (Factor V Leiden [FVL] and G20210A F2 and C46T F12). The sequences are detected by enzyme-linked immunosorbent assay (ELISA)-resolved colorimeter after hybridization with an amplification product labeled with digoxygenin. To evaluate the reliability of this method, we analyzed 122 deoxyribonucleic acid (DNA) samples of known genotypes for these three mutations. Six subjects initially assigned as heterozygous for FVL and two subjects assigned as normal for the G20210A F2 mutation showed discrepancies between the current techniques and our newly-developed ELISA-based technique. When these samples were sequenced the concordance using our method was 100%. Thus, initially they were assigned incorrectly based on the available methodologies. It is noteworthy that our method is adaptable to an Automated ELISA Analyzer that allows for routine processing of both small and large numbers of DNA samples. We present a robust, rapid, reproducible, cost-effective, and simple multiplex PCR ELISA method to simultaneously detect carriers of thrombotic genetic risk factors. Testing for thrombophilia should contribute to better diagnosis, prevention, and treatment strategies, providing valuable information to assess the risk of recurrence in the proband, and in family members who are asymptomatic.