Stereotactic biopsies, which are widely used in neurosurgery for tissue sampling and brain lesion diagnosis, are preferred for deep lesions because of their minimal invasiveness and precision, reducing the morbidity associated with open biopsies. This is especially relevant for brainstem tumors, which have been historically treated without histological confirmation due to concerns over morbidity and mortality. Recent advancements in the technique and understanding of brainstem tumor biology have altered treatment protocols. This chapter demonstrates that stereotactic biopsy is an effective diagnostic tool for brainstem lesions in both adults and children. Although it carries higher functional and fatal risks than biopsies of the brain or cerebellum, its safety profile remains acceptable. Thus, the benefit-risk ratio is favorable, but should be evaluated individually. Owing to the rarity of biopsy-amenable brainstem lesions, clinicians should confirm the biopsy indication through a specialized multidisciplinary group and refer patients to a neurosurgical team experienced in brainstem biopsies.