Computer-assisted procedures are used to measure infarct areas in animal stroke models, but this approach usually follows the less objective manual tracing of the boundaries of the infarct. Building on previously reported methodology using scanned images of triphenyltetrazolium chloride (TTC)-stained rat brains in the intraluminal thread model, we developed an objective method to assess ischemic damage in both the mouse and rat brains. The unique addition to our approach is the use of sham-treated animals, which thereby permits the removal of normal brain white matter from the ipsilateral injured brain. All brain sections per animal were scanned simultaneously using a Microtek Scanmaker 4 flatbed scanner. Color segmentation on full color images of 2 mm coronal brain sections was performed. Using Image Pro Plus (4.0) and color segmentation, ischemic and normal white matter areas were measured in the green channel and the entire brain area in the red channel. The percent of unstained tissue was calculated for sham-treated animals and for those with cerebral ischemia. By subtracting the average unstained area of the sham-treated group from the average unstained area from the ischemic group, the ischemic area was calculated. This methodology was validated using mouse and rat permanent and transient, focal ischemia models and MK-801 in the permanent ischemia models. MK-801, dosed at 3 mg/kg i.p. prior to the injury, reduced the injury by 75% in the mouse and 44% in the rat permanent occlusion models. The benefits of this methodology include: objectivity of the analysis of the ischemic injury, use of readily available software so that costs can be contained and removal of normal subcortical white matter from the calculation. This method should allow more consistent evaluation of changes in the infarct size, therefore, resulting in reduced variability and higher productivity.