HBC and polybrene neutralized heparins and enabled monitoring of anticoagulant activity of dabigatran in the TT test. In these tests, plasma with dabigatran or rivaroxaban was mixed with UFH or enoxaparin and then incubated with HBC or polybrene, respectively. The thrombin time (TT) and anti-factor Xa activity were monitored in pooled plasma from healthy volunteers. Using the heparin-binding copolymer (HBC) and polybrene, we aimed to develop a solution for monitoring the anticoagulant activity of DOACs in human plasma in the interfering presence of unfractionated heparin (UFH) and enoxaparin. The routine monitoring of direct oral anticoagulants (DOACs) may be considered in patients with renal impairment, patients who are heavily obese, or patients requiring elective surgery. HBC administered in an appropriate dose might be an efficient substitute for PS to reverse significantly increased anticoagulant activity that may be connected with major bleeding in patients receiving ENX subcutaneously. Histopathological analysis showed changes in the liver, lungs, and spleen of mice treated with HBC and in the lungs and heart of mice treated with PS. PS did not reverse antifactor Xa activity and partially reversed antifactor IIa activity. Both doses of HBC completely reversed the effect of ENX on antifactor IIa activity. HBC at the lower dose reversed the effect of ENX on antifactor Xa activity for 10 min after antidote administration, whereas at the higher dose, HBC reversed the effect on antifactor Xa activity throughout the course of the experiment. The main organs were collected for histological analysis. The activities of antifactors Xa and IIa and biochemical parameters were measured. The blood was collected after 3, 10, 60, 120, 360, and 600 min after vehicle, HBC, or PS administration. After 110 min, vehicle, HBC (6.25 and 12.5 mg/kg), or PS (5 and 10 mg/kg) were administered into the tail vein. BALB/c mice were subcutaneously injected with ENX at the dose of 5 mg/kg. Here, we focused on the HBC inhibitory activity against subcutaneously administered ENX in healthy mice. We developed a diblock copolymer, heparin-binding copolymer (HBC), that reverses intravenously administered heparins. The only registered antidote for ENX, protamine sulfate (PS), has 60% efficacy and can cause severe adverse side effects. Uncontrolled bleeding after enoxaparin (ENX) is rare but may be life-threatening.