Background: Radiotherapy (RT) is recommended to all patients undergoing Breast Conserving Surgery (BCS). Two strategies can be applied to irradiation, External Beam RT (EBRT) in addition, Intraoperative Radiation Therapy (IORT). The aim of this study was to introduce a protein biomarker panel related to molecular function of IORT. Materials and Methods: Six Breast Cancer (BC) patients as a pilot study were treated by 12 Gy (Boost dose) and 21 Gy (Radical dose). Samples tissue included Margin before IORT (MB), and Margin 24 hours After IORT (MA24 h). High-throughput technology such as Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) was performed to study proteomic of IORT-treated tumor bed. Results: We classified 110 differentially expressed proteins (DEPs) as a protein biomarker panel by mapping the annotated coding region sequences to the reference canonical pathways in the KEGG database. Conclusion: The analyses of protein function and signaling pathways of these DEPs reveal a molecular response to the effectiveness of IORT.