Background: High-grade gliomas (HGG) are malignant brain tumors associated with frequent recurrent disease. Clinical management of HGG patients is currently devoid of blood biomarkers for early diagnosis, monitoring therapeutic effects and predicting recurrence. Different circulating miRNAs, both free and associated with exosomes, are described in patients with HGG. We previously identified miR-21, miR-222 and miR-124-3p purified from serum exosomes as molecular signature to help pre-operative clinical diagnosis and grading of gliomas. The aim of the present study was to verify this signature as a tool to assess the effect of treatment and for the early identification of progression in newly diagnosed HGG patients. Material and Methods: Major inclusion criteria were newly diagnosed, histologically confirmed HGG patients, no prior chemotherapy, ECOG PS 0-2 and patients scheduled for radiochemotherapy with temozolomide as first-line treatment after surgery. RANO criteria were used for response assessment. Serum was collected at baseline and subsequently at each neuroradiological assessment. mir-21, -222 and -124-3p expression in serum exosomes was measured in all samples. Results: A total number of 57 patients were enrolled; 41 were male, 52 with glioblastoma and 5 with anaplastic astrocytoma; 18 received radical surgery. HGG patients with higher exosomal miRNA expression displayed a statistically significant lower progression-free survival and overall survival. Increased expression of miR-21, -222 and -124-3p during post-operative follow-up was associated with HGG progression. Conclusions: These data indicate that miR-21, -222 and -124-3p in serum exosomes may be useful molecular biomarkers for complementing clinical evaluation of early tumor progression during post-surgical therapy in patients with HGG.