Background: Frameless stereotactic biopsy represents a minimally invasive procedure used for the histopathological diagnosis of brain tumors or to safely approach deep-seated lesions near eloquent areas not amenable for classical neurosurgical procedures. Traditionally, biopsy is performed relying on anatomical landmarks, but it can lead itself to intra- and postoperative complications, such as hemorrhage and fiber disruption. Diffusion tensor imaging (DTI) tractography represents a useful tool that can analyze the individual fiber tract conformation in cases of brain tumor and consequently identify the best biopsy trajectory, preserving white matter pathways. In our study, we present a novel technique that is based on the use of preoperative DTI for biopsy. Methods: Between January 2018 and January 2020, data about patients who underwent frameless biopsy using DTI tractography were retrospectively reviewed. The inclusion criterion was adult patients eligible for elective surgery for a single or multiple deep-seated lesions with contraindications to complete surgical resection. Results: We included 12 patients (mean age of 67.9 [±9.6] years). A single cranial lesion was detected in 7 cases, and multiple lesions in 5 cases. The use of DTI enabled the identification of white matter pathways in all cases and adjustment of the biopsy trajectory based on anatomical landmarks in 7 cases. Postoperative hematoma was reported in 1 case, and histological diagnosis was obtained in 11 cases. Conclusion: According to our results, tractography is a useful tool that can enhance the safety of cerebral lesions biopsy sparing any fiber tract damages. © 2020 Elsevier Inc.