Thrombin is a coagulation factor implicated in various pathological and physiological processes in the brain, exerting beneficial and deleterious effects in a concentration-dependent manner. Measurement of thrombin activity levels in pathological animal models is needed and in some cases, because of technical considerations, only frozen samples are available. In the current study, we used a quantitative method to evaluate thrombin activity in fresh and frozen brain sections of 43 male and female adult healthy mice. We stratified data per brain section, brain hemisphere, and mouse sex. We found lower thrombin activity in frozen sections compared with fresh sections, falling within levels considered central nervous system protective in previous studies. The results suggest that fresh section thrombin activity levels in healthy mice can be extrapolated from frozen brain sections. In addition, we found varying thrombin activity across the brain sections, with maximal activity in the olfactory system and hippocampus-containing sections. Thrombin activity did not vary between males and females, or between the right and the left hemispheres, in a statistically significantly manner.