Standardization of pre-analytical conditions is the obligatory step for all potential diagnostic tests. Spatial clot growth (Thrombodynamics) is a new global hemostasis assay that considers spatial organization of coagulation. The principal parameter is rate of fibrin clot growth from the tissue-factor coated surface. In this work we studied the pre-analytical variables of Thrombodynamics assay that include conditions of blood collection, sample preparation and storage.Blood of apparently healthy volunteers was used. Eight types of citrate blood collection tubes were tested, centrifugation conditions for plasma preparation were evaluated and impact of plasma freezing/thawing was tested.Among the blood collection tubes tested, BD Vacutainer glass tubes showed a significantly higher clot growth rate compared to plastic tubes. There was no difference between 3.2% and 3.8% of sodium citrate. For plasma preparation, a single 15min centrifugation at 1 600g shows significantly increased clot growth rate compared to plasma obtained by two sequential centrifugations (15min 1 600g, 5min 10 000g). There was no significant difference between 1 600g and 2 100g if the second centrifugation was performed. For the second centrifugation there was no difference between 20min at 1 600g and 5min at 10 000g. Frozen-thawed plasma showed increased clot growth rate compared to fresh plasma.The data represent the necessary steps for the standardization of Thrombodynamics assay and for the formulation of the operating guide.