The most common lesions located in the sphenoidal, sellar, parasellar, and suprasellar areas, such as pituitary adenomas, Rathke’s cleft cysts, craniopharyngiomas, chordomas, or chondrosarcomas, usually have a well-identified MRI presentation and a well-known course, with a well-defined consensus management leading to direct surgical resection in most cases (Melmed, The pituitary, 4th edn. Elsevier/Academic Press, London/San Diego, 2017; Buchfelder et al., Pituitary 16:18–25, 2013; Wang et al., Int Forum Allergy Rhinol 9:S145–S365, 2019; Henderson and Schwartz, J Neuro-Oncol 156:97–108, 2022). However, other tumoral, inflammatory, granulomatous, or infectious lesions may have a more atypical clinical and radiological presentations, justifying surgical biopsy as the first step in therapeutic management (Hána et al., Neuroendocrinology 110:809–821, 2020; Devuyst et al., Eur J Endocrinol 181:95–105, 2020). Given the complexity of skull base anatomy, the multiplicity of surgical approaches, and the high number of pathologies involved, the strategy of biopsies in the sphenoidal area or in the hypothalamo-pituitary tract remains challenging for the neurosurgeon (Wang et al., Int Forum Allergy Rhinol 9:S145–S365, 2019; Baussart et al., Acta Neurochir 163:655–659, 2021; Frara et al., Endocrinol Metab Clin N Am 49:553–564, 2020). Lesions involving the sphenoid area or hypothalamo-pituitary tract are close to highly functional vascular or neurological structures, explaining why the decision to perform a surgical biopsy should be taken after multidisciplinary discussion in centers of expertise. Indication is based on a rigorous analysis of the clinical history, preoperative MRI, and hormonal and biological status. In this chapter, we propose a reliable strategy for surgical biopsy in this eloquent area, focusing on indication, anatomical location, pathologies, and specific surgical approaches.