Aims: Functional magnetic resonance imaging (fMRI) processing may vary depending on the pre-processing steps, analysis strategy, thresholding and, therefore, the software itself. Pre-processing has been optimized in the pipeline fMRIPrep; however, the pipeline does not have a clinician-friendly interface and requires additional analysis steps to be performed elsewhere. We compare the results of language lateralization from fMRI when processed start-to-finish using two different popular open-source software packages, SPM12 and FSL or when preprocessed with fMRIPrep.Methods: Patients with epilepsy (n=12) and healthy controls (n=12) participated in two task-based fMRI paradigms; sentence completion and word generation. Pre-processing steps include motion correction, coregistration, segmentation, normalization, and smoothing. The analysis includes general linear model (GLM) and laterality index (LI) calculations using tools specific to each software. Laterality indices were computed for three language regions of interest (ROIs). A total of 144 LIs were calculated (24 subjects * 2 tasks * 3 ROI/task) for each of the 4 processing pipelines. Overlap of activations was assessed visually and with Dice coefficients.Results: Visual assessment of overlap showed similar results, but Dice scores ranged from 0.16-0.71 due to differences in distortion correction. However, LI remained concordant (showing the same language lateralization) in 531/576 (92%) of cases.Conclusions: These findings indicate that all tools perform similarly for block design language fMRI data. The choice of software for clinical use ultimately depends on factors such as usability and alignment with the standard practices of individual epilepsy surgery centers.Funding: Queen’s University Faculty of Health Sciences