Background <p>Brain metastases have traditionally been considered well-demarcated lesions; however, increasing evidence demonstrates frequent microscopic infiltration of the surrounding brain parenchyma, with relevant prognostic implications, despite current intraoperative tools. The aim of this study was to evaluate the feasibility and diagnostic performance of a label-free nanoplasmonic biosensor for intraoperative discrimination between tumor tissue and peritumoral brain in brain metastases surgery.</p> Methods <p>A prospective multicenter study was conducted in patients undergoing surgical resection of brain metastases. Paired tumor and adjacent peritumoral tissue samples were collected intraoperatively following a standardized protocol across participating centers. Samples were analyzed ex vivo using a plasmonic nanostructured biosensor, which detects tissue-specific refractive index differences. Histopathological examination served as the reference standard. Paired comparisons were performed using the Wilcoxon signed-rank test, and diagnostic performance was assessed using receiver operating characteristic analysis.</p> Results <p>Twenty paired tumor and peritumoral samples from a consecutive series of 20 patients were analyzed. Refractive index values were significantly higher in tumor tissue compared with peritumoral brain (<i>p</i> = 0.0008). In 85% of cases, tumor samples showed higher refractive index values than their paired peritumoral counterparts. Using the optimal cut-off value, sensitivity was 76% and specificity was 68%.</p> Conclusion <p>Tumor and peritumoral brain tissue can be discriminated through the measurement of intrinsic biophysical properties with a label-free nanoplasmonic biosensor, supporting its potential role as an objective intraoperative tool for margin assessment without the need of exogenous agents.</p>