Laser speckle flowmetry (LSF) can measure moving red blood cells in real time as changes in tissue blood flow; however, LSF values may not be sufficiently reproducible due to body movements and other factors. Therefore, it is difficult to compare absolute values of LSF directly. This study aimed to verify the influence of head fixation on improving the measurement accuracy and enabling the comparison of LSF values semi-quantitatively using an external standard. We first examined the effects of head fixation on LSF values obtained during cerebral blood flow (CBF) measurement through a bone window over the cerebrum in Sprague-Dawley rats. The LSF values in the bone window were semi-quantitated using the ratio of the external standard LSF values measured simultaneously in the same field of view in a unilateral common carotid artery ligation model to evaluate CBF. Head fixation halved the variability of LSF values when compared with measurements made without head fixation. The LSF laser focus position did not affect the LSF values provided the focus was positioned within the exposed cranium. Semi-quantitation of LSF values made it possible to monitor changes in CBF in each respective bone window over time in a model of unilateral carotid ligation. In conclusion, the stabilizing effect of head fixation on LSF values was clarified, and we observed that to improve measurement accuracy, the head should be immobilized. Semi-quantitation of LSF values was first introduced by using an external standard and appears to be useful in monitoring the CBF of each location separately.