Background: Traumatic brain injury (TBI) during birth constitutes one of the most relevant causes of mortality and morbidity in newborns worldwide. Although improvements in obstetrical management and better indications for caesarean section have led to a consistent decrease in the incidence of perinatal mechanical injury, vacuum extraction is still associated with a high complications rate leading to several forensic issues in the evaluation of healthcare professional management. Methods: Vacuum-associated lesions may be topographically distinguished as extracranial or intracranial injuries. In order to achieve a correct assessment, diagnostic procedure should include post-mortem computed tomography and magnetic resonance imaging, autopsy examination, brain sampling and histological/immunohistochemical examination. Results: Post-mortem imaging represents a valid aid to guarantee preliminary evidence and direct subsequent investigations. An appropriate autopsy sampling must include several areas of cortex and underlying white matter; moreover, any visceral hemorrhages or other lesions should be sampled for the histological and immunohistochemical assessment of vitality and timing. Conclusions: This study aimed to promote a validated step-by-step procedure to be adopted in order to standardize and to make easier the post-mortem framing and timing of vacuum-associated pediatric brain injuries.