Despite the clinical use of well-established and newer anticoagulant drugs, venous thromboembolism (VTE) remains a major public health problem associated with high morbidity and mortality. The search for new anticoagulant drugs with an improved benefit/risk ratio is therefore a main challenge for the pharmaceutical industries. Success in this endeavour is driven by the right completion of the preclinical studies. In this work, a series of relevant and reliable pharmacological tools modeling thrombotic disorders was successfully developed and validated to evaluate prospective anticoagulant compounds. This preclinical platform consisted in three drug testing levels: (1) in vitro enzymatic assays on isolated coagulation factors (i.e. thrombin, factor Xa, tissue factor/factor VIIa complex and factor XIIa); (2) functional ex vivo thrombin generation assays in human plasma and (3) an in vivo venous thrombosis model induced by stasis and endothelial damages and coupled to thrombin generation assay in the rat. This preclinical testing platform was developed and evaluated during the evaluation of chemically distinct compounds selected for their potential anticoagulant and antithrombotic properties. Three types of products were investigated in order to identify promising innovative anticoagulants for the prevention and treatment of VTE: coumarins, the tick protein Ixodes ricinus contact phase inhibitor (Ir-CPI) and leaf extracts/fractions from Croton zambesicus.