fXa inhibitors are a class of anticoagulant drugs widely prescribed to prevent strokes, venous thromboembolism, and deep vein thrombosis. The major concern for patients using this class of drugs is an increased risk of major bleeding events. Andexanet alfa (AnXa) is a modified fXa protein that has a high affinity for fXa inhibitors. When patients treated with fXa inhibitors are exposed to AnXa the unbound plasma concentration of the fXa inhibitors are sequestered and coagulation is restored. In order to establish the effectiveness of AnXa the unbound concentration of the fXa inhibitors must be determined. In this work, we report the validation of four fXa inhibitors in human plasma by HPLC-MS/MS used to support multiple AnXa clinical studies.