Atlas and fMRI-based approaches to standardise seed and end regions for probabilistic tractography of the arcuate fasciculus are investigated. fMRI-based approaches use spheres centered on language task peak activation. Within this pilot cohort, an atlas-based approach demonstrates the greatest sensitivity. fMRI-based approaches are more specific, but sensitivity can be increased by enlarging sphere size. Within each approach, a trade-off between sensitivity and specificity is seen as seed/end region size increases. For patients with abnormalities or lesions, where atlas approaches might be compromised, fMRI methods may be preferred. Further work to optimise fMRI-based approach is warranted, alongside application to patient data.