Brainstem tumors account for 10-20% of pediatric brain tumors with a peak age of diagnosis between 7 and 9 years old and are often fatal. Historically, diagnosis of brainstem tumors has been largely based on imaging; however, recent studies have demonstrated the incongruities between preoperative MRI diagnosis and postoperative pathological findings highlighting the importance of brainstem biopsy for diagnostic accuracy. Stereotactic brainstem biopsy for pediatric brainstem tumors has been proven to be safe with a high diagnostic yield (96.1-97.4%) and relatively low morbidity and mortality. Successful pediatric brainstem tumor biopsy demands intricate knowledge of brainstem anatomy, cranial nerves and vasculature, and common pediatric brainstem tumors by the performing surgeon. Additionally, understanding of the surgical indications and techniques (e.g., frame-based versus frameless, robotic assistance, surgical approach, and targets selection) helps to ensure maximal safety and tissue yield. Pediatric brainstem biopsy permits histological conformation of brainstem lesions leading to accurate diagnosis and the potential for personalized treatment and future therapeutic research.