Radiotherapy induces systemic changes beyond the targeted tumour site, yet the biological mechanisms driving these effects remain poorly understood. Here, we apply the Nano-proteomics pipeline to longitudinally profile systemic responses to radiotherapy in patients with prostate, bladder, and head and neck cancers. Weekly plasma samples collected before and during treatment were analysed by mass spectrometry and revealed acute plasma proteomic changes. The most significant systemic effects occurred within the first two weeks of radiotherapy, highlighting a critical window for biomarker discovery. Across all cohorts, common biological processes were enriched, characterized by early activation of inflammatory and immune pathways, followed by later structural reorganization and immune resolution, regardless of irradiated tissue or concurrent treatments. Despite this shared acute response, distinct protein mediators were dysregulated in a tumour-specific manner. Notably, in prostate cancer patients, baseline proteomics revealed 28 biomarkers associated with late bowel and urinary toxicities, supporting the potential of plasma proteomic biomarkers to identify patients at increased risk of radiotherapy-induced toxicity. This study underscores the value of longitudinal proteomics in uncovering systemic effects of radiotherapy and paves the way for developing predictive biomarkers to guide personalized radiotherapy strategies aimed at minimizing adverse effects.