Accurate pre-clinical study reporting requires validated processing tools to increase data reproducibility within and between laboratories. Segmentation of rodent brain from non-brain tissue is an important first step in preclinical imaging pipelines for which well validated tools are still under development. The current study aims to clarify the best approach to automatic brain extraction for studies in the immature rat. Skull stripping modules from AFNI, PCNN-3D, and RATS software packages were assessed for their ability to accurately segment brain from non-brain by comparison to manual segmentation. Comparison was performed using Dice coefficient of similarity. Results showed that the RATS package outperformed the others by including a lower percentage of false positive, non-brain voxels in the brain mask. However, AFNI resulted in a lower percentage of false negative voxels. Although the automatic approaches for brain segmentation significantly facilitate the data stream process, the current study findings suggest that the task of rodent brain segmentation from T2 weighted MRI needs to be accompanied by a supervised quality control step when developmental brain imaging studies were targeted.