The Total Thrombus-formation Analyser System (T-TAS) is a whole blood flow chamber system for the measurement of in vitro thrombus formation under variable shear stress conditions. Our current study sought to evaluate the potential utility of the T-TAS for the measurement of thrombus formation within human and mouse whole blood. T-TAS microchips (collagen, PL chip; collagen/tissue thromboplastin, AR chip) were used to analyse platelet or fibrin-rich thrombus formation respectively. Blood samples from humans (healthy and patients with mild bleeding disorders) and wild-type (WT), mice were tested. Light transmission lumi-aggregometer (lumi-LTA) was performed in PRP using several concentrations of ADP, adrenaline, arachidonic acid, collagen, PAR-1 peptide and ristocetin. Thrombus growth (N=22) increased with shear within PL (4:40±1.11, 3:25±0.43 and 3:12±0.48 mins [1000, 1500 and 2000s-1]) and AR chips (3:55±0.42 and 1:49±0.19 [240s-1 and 600s-1]). The area under the curve (AUC) on the PL chip was also reduced at 1000s-1 compared to 1500/2000s-1 (260±51.7, 317± 55.4 and 301±66.2 respectively). In contrast, no differences in the AUC between 240s-1 and 600s-1 were observed in the AR chip (1593±122 and 1591±158). The intra-assay coefficient of variation (CV) (n=10) in the PL chip (1000s-1) and AR chip (240s-1) were T1014.1%, T6016.7%, T10-6022.8% and AUC1024.4% or T10 9.03%, T808.64%, T10-8023.8% and AUC305.1%. AR chip thrombus formation was inhibited by rivaroxaban (1µM), but not with ticagrelor (10µM). In contrast, PL chip thrombus formation was totally inhibited by ticagrelor. T-TAS shows an overall agreement with lumi-LTA in 87% of patients (n=30) with normal platelet counts recruited into the genotyping and phenotyping of platelet (GAPP) study and suspected to have a platelet function defect. The onset (T10) of thrombus formation in WT mice (N= 4) was shorter when compared to humans e.g. PL chip (1000s-1) T10 were 02:02±00:23 and 03:30±0:45 respectively). T-TAS measures in vitro thrombus formation and can be used for monitoring antithrombotic therapy, investigating patients with suspected platelet function defects and monitoring platelet function within mice.