Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. The long-term cŽmƉůŝcĂƟŽnƐ arising from standard therapy with surgery, chemotherapy and radiotherapy lower the quality of life (QOL) due to ĞĂƟnŐ disorders, speech problems and cŽƐmĞƟc issues. Recently the importance of ĂnƟbŽĚy therapy has increasingly come to be recognized for its capacity to enhance QOL. Robo1, an axon guidance receptor, has been received considerable ĂƩĞnƟŽn as a monoclonal ĂnƟbŽĚy target in various cancers. Methods and ĮnĚŝnŐƐ͗ We checked the expression of Robo1 and the cytotoxic ĞīĞcƚ of saporin-conjugated ĂnƟͲ RŽbŽ1 immunotoxin (IT) against several HNSCC cell lines. The Robo1 cell surface expression level ĞƐƟmĂƚĞĚ by ŇŽw cytometry using the ĂnƟbŽĚy B5209B was approximately 220,000 copies in Robo1 over-expressed CHO (Robo1/ CHO) cells, and in HNSCCs, 22,000 copies in HSQ-89 cells, 3,000 copies in Sa3 cells, and few copies inSAS cells. CŽnvĞnƟŽnĂů IT treatment exhibited an inadequate cytotoxic ĞīĞcƚ even in HSQ-89 cells. When we added a ƉŚŽƚŽƐĞnƐŝƟnjĞr and LED light ŝůůƵmŝnĂƟŽn (650 nm), the cytotoxic ĞīĞcƚ was remarkably improved in HSQ89. With longer exposure, ƐŝŐnŝĮcĂnƚ cytotoxicity was observed, even in Sa3 cells. Conclusion: These results suggest that the photochemical ŝnƚĞrnĂůŝnjĂƟŽn (PCI) is a promising method for ĂƵŐmĞnƟnŐ the cytotoxicity of IT against tumors. The drug delivery system shown in this study should be applicable to other targets with low level expression on cancer cells, thus widening the ƚŚĞrĂƉĞƵƟc window in rare cancers.