Unfractionated heparin (UFH) has long been an anticoagulant of choice. Despite the increasing use of low molecular weight heparin derivatives and more recently direct oral anticoagulants, UFH is still prescribed to prevent or treat thrombosis in specific populations at high risk of thrombosis or bleeding, including patients with severe renal impairment, critically ill patients, or those undergoing cardiac catheterization/surgery. Due to its narrow therapeutic index, with a risk of hemorrhage in case of overdosing and a risk of thrombosis in case of underdosing, monitoring its anticoagulant activity is considered standard of care even though scientific data remains limited. Despite being used for decades, some unanswered questions remain, particularly regarding the biological monitoring of its efficacy. Which assay can be used? In practice, the most widely used tests are plasma-based, such as the aPTT, a clotting assay, or the anti-Xa activity, a chromogenic substrate-based assay, whereas whole blood tests such as the ACT are limited to specific situation such as extracorporeal circulation. Which therapeutic ranges should be used? An aPTT prolongation of 1.5–2.5 × control, or an anti-Xa activity of 0.30–0.70 IU/mL, corresponding to 0.2–0.4 U/mL by protamine titration, ranges are commonly used, despite limited evidence-based data. Furthermore, these assays are not standardized due to some degree of heterogeneity in the sensitivity of reagents to UFH. Moreover, some pre-analytical questions remain, particularly regarding the collection tube to be used for example, citrate or CTAD anticoagulant solution, full- or partial-draw (half-filled) tubes, and the maximum time between collection and analysis.