Anticoagulation therapy is a mainstay of the treatment of thrombotic disorders; however, conventional anticoagulants trade antithrombotic benefits for bleeding risk. Factor (f) XI deficiency, known as hemophilia C, rarely causes spontaneous bleeding, suggesting that fXI plays a limited role in hemostasis. In contrast, individuals with congenital fXI deficiency display a reduced incidence of ischemic stroke and venous thromboembolism, indicating that fXI plays a role in thrombosis. For these reasons, there is intense interest in pursuing fXI/factor XIa (fXIa) as targets for achieving antithrombotic benefit with reduced bleeding risk. To obtain selective inhibitors of fXIa, we employed libraries of natural and unnatural amino acids to profile fXIa substrate preferences. We developed chemical tools for investigating fXIa activity, such as substrates, inhibitors, and activity-based probes (ABPs). Finally, we demonstrated that our ABP selectively labels fXIa in the human plasma, making this tool suitable for further studies on the role of fXIa in biological samples.