Thrombophilia is a common concept that describes an elevated tendency and associated morbidity for thrombosis. The prevalence of genetic risk factors for thrombosis varies widely around the world. For the last 25 years, risk factors for venous thromboembolism (VTE) have been related to mutations in the genes of the coagulation/anticoagulation system. In the majority of cases of inherited thrombophilia, a single nucleotide polymorphism in the gene that codes for coagulation factor II (prothrombin G20210A) has been involved and thus represents the most frequently asked investigation of risk factors predisposing to VTE and inherited thrombophilia. The determination of this mutation should be part of the main examination of patients with unexplained thrombosis. Tests to diagnose this mutation are conducted to examine a personal or family history of VTE. There are currently several different approaches available for the identification of this mutation in the laboratory. This chapter aims to discuss effective assessments for detecting the G20210A prothrombin mutation, applicability, benefits, and drawbacks of use as expressed in the evidence published and the consensus opinion of recognized experts in the field.