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Background: High-Grade Gliomas are the most common primary brain malignancies and despite the multimodal treatment, and the increasing amount of adjuvant treatment options the overall prognosis remains dismal. The present investigation aims to analyze the safety profile of the use of intraoperative ultrasounds (Io-US) in a homogeneous and matched cohort of patients suffering from High-grade gliomas (HGG) operated on with or without the aid of Io-US and Fluorescein in specific relation to the incidence of neurological and functional status sequelae., Methods and Materials: A retrospective analysis was performed on 74 patients affected by HGG. 22 patients were treated with Io-US matched with neuronavigational system (Group A); 15 patients were treated both with the use of Io-US and Fluorescein matched with neuronavigational system (Group B); 37 patients were treated with the use of the neuronavigational system only (Group C). Primary endpoints were the extent of resection and functional outcome (measured with Karnofski Performance Status)., Results: Significative differences were observed in terms of a higher extent of resection in Group B. In a multivariate analysis, this data appears to be independent of the location (eloquent/non-eloquent) of the lesion and from its histology. Regarding functional outcomes, no differences were detected between the two groups., Conclusions: The present study is the first that analyzes the simultaneous use of Io-US and Fluorescein, and the results demonstrate that these two instruments together could improve the extent of resection in HGG while ensuring good outcomes in terms of functional status., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)