Presurgical determination of language laterality is an important step for assessing potential risk of dysfunction resulting from brain resection within or near suspected language areas. Image-based functional MRI (fMRI) methods seek to address limitations to the clinical gold-standard technique by offering a safer, less costly, and non-invasive alternative. In this study we outline a set of protocols for objective determination of langue-specific asymmetry from fMRI activation maps. We studied 13 healthy, right-handed volunteers using a vocalized antonym-generation task. Initially, using the standard threshold-dependent laterality index (LI) procedure, we demonstrated an undesirably high degree of intra-subject variability and indeterminacy in LI value. We addressed this issue by implementing a novel threshold-independent method, resulting in a single, unambiguous LI for each subject. These LIs were then averaged across the group and used to compare functional laterality within the whole hemispheric volumes and six intra-hemispheric regions-of-interest (ROIs). We noted that as a result of increased bilateral activation from vocalizations, laterality assessment calculated from the whole hemisphere resulted in insignificant asymmetry. However, by focusing the LI exclusively on the inferior frontal (IFG) and supramarginal gyri (SMG), robust leftward asymmetries were observed. We also examined the influence of stimulus mode on the group mean ROI LI, and observed an increase in IFG asymmetry using visual mode, and in SMG using the auditory mode. Based on these findings, we make recommendations for optimized presurgical protocols.