Atrial fibrillation (AF) catheter ablation is performed in patients receiving direct oral anticoagulants (DOACs) with intra-procedural unfractionated heparin (UFH) administration to achieve activated clotting time (ACT) at 300 s, as for vitamin K antagonist (VKA). We determined whether ACT monitoring might be transposed from VKA to DOAC-treated patients. Blood was taken from 124 patients receiving uninterrupted dabigatran, rivaroxaban, apixaban, or VKA or being untreated. DOAC concentration or INR (VKA) were measured. ACT was determined at baseline, and after spiking with UFH doses equivalent to 1000, 2500, 5000 and 10000 IU in vivo. At baseline, anticoagulants prolonged ACT differently, ACT was longer with dabigatran and shorter with apixaban despite similar concentrations. ACT strongly correlated with INR and dabigatran concentration, but not with apixaban or rivaroxaban concentrations. Moreover, UFH effects on ACT prolongation depended on the anticoagulant: dose-response curves in samples with VKA and dabigatran were parallel whereas ACT prolongation in response to UFH was significantly smaller with rivaroxaban and especially apixaban. Therefore, UFH to achieve ACT at 300 s might be transposed from VKA to uninterrupted dabigatran-treated patients but not to patients receiving FXa-inhibitors, especially apixaban. Targeting 300 s might expose to UFH overdosing and bleeding, questioning the current anticoagulation strategy.