Radiotherapy has an important role in the treatment of brain metastases (BrM) but carries risk of short and/or long-term toxicity, termed radiation-induced neurotoxicity (RIN). As the diagnosis of RIN is crucial for correct pa-tient management, there is an unmet need for reliable biomarkers for RIN. The aim of this proof-of concept study was to determine the utility of brain-derived circulating free DNA (BncfDNA), identified by specific methylation patterns for neurons, astrocytes, and oligodendrocytes, as RIN biomarkers. Twenty-four patients with BrM were monitored clinically and radiologically before, during and after brain radiotherapy, and blood for BncfDNA analysis (98 samples) was concurrently collected. Sixteen patients were treated with whole brain radiotherapy and eight patients with stereotactic radiosurgery. During follow-up nine RIN events were detected, and all correlated with significant increase in BncfDNA levels compared to baseline. Additionally, resolution of RIN correlated with de-crease in BncfDNA. Changes in BncfDNA were independent of tumor response. Elevated BncfDNA levels reflects brain cell injury incurred by radiotherapy, further research is needed to establish BncfDNA as a novel plasma-based biomarker for RIN.