In glioma surgery, maximizing the extent of resection while preserving cognitive functions requires an understanding of the unique architecture of the white matter (WM) pathways of the single patient and of their spatial relationship with the tumor. Tractography enables the reconstruction of WM pathways, and bundle segmentation allows the identification of critical connections for functional preservation. This study evaluates the effectiveness of a streamline-based approach for bundle segmentation on a clinical dataset as compared to the traditional ROI-based approach. We performed bundle segmentation of the arcuate fasciculus, of its indirect anterior and posterior segments, and of the inferior fronto-occipital fasciculus in the healthy hemisphere of 25 high-grade glioma patients using both ROI- and streamline-based approaches. ROI-based segmentation involved manually delineating ROIs on MR anatomical images in Trackvis (V0.6.2.1). Streamline-based segmentations were performed in Tractome, which integrates clustering algorithms with the visual inspection and manipulation of streamlines. Shape analysis was conducted on each bundle. A paired -test was performed on the irregularity measurement to compare segmentations achieved with the two approaches. Qualitative differences were evaluated through visual inspection. Streamline-based segmentation consistently yielded significantly lower irregularity scores ( < 0.001) compared to ROI-based segmentation for all the examined bundles, indicating more compact and accurate bundle reconstructions. Qualitative assessment identified common biases in ROI-based segmentations, such as the inclusion of anatomically implausible streamlines or streamlines with undesired trajectories. Streamline-based bundle segmentation with Tractome provides reliable and more accurate reconstructions compared to the ROI-based approach. By directly manipulating streamlines rather than relying on voxel-based ROI delineations, Tractome allows us to discern and discard implausible or undesired streamlines and to identify the course of WM bundles even when the anatomy is distorted by the lesion. These features make Tractome a robust tool for bundle segmentation in clinical contexts.