Protamine sulfate (protamine), a low molecular weight poly-cationic amine, has been used for some time on patients undergoing cardiac operations and procedures. It is particularly used on patients who have been treated with heparin (an anticoagulant), in order to neutralize the anticoagulant effects of heparin on the person's blood. In this in vitro study, various 500 μL one-day old citrated whole human blood samples (n=18) have been subjected to different amounts of protamine of a single concentration (10 mg/mL in saline), using 0.9% saline added to blood as control. It is known that saline, by itself, has no effect on blood clotting. After thorough mixing and incubation for 10 min. at 37°C, the time it takes for 200 μL of each sample to clot, in presence of 20 μL 0.1 M CaCl2 was measured. The results show that protamine acts as an anticoagulant in the absence of heparin. Clotting time also increases with more protamine added.