In order to identify membrane proteins whose expression is induced by X-ray irradiation, we developed an antibody (Ab)-directed strategy using a phage Ab library. X-ray-irradiated cells were screened with a phage Ab library in the presence of a large excess of polyclonal Abs prepared against membrane proteins that are commonly present at the surface of both X-ray-irradiated and non-irradiated cells. After isolation of Ab that bound only to X-ray-irradiated cells, the Ag was identified using mass spectrometry. Using this approach, we found that expression of LY6D is induced in MCF10A cells by X-ray irradiation. The induction of LY6D expression is triggered through a pathway regulated by ATM, CHK2, and p53. This method is a new Ab-directed proteomic strategy for analysis of membrane proteins, and is applicable to various biological phenomena in situations in which both target molecule-expressing cells and non-expressing cells are available. © 2012 The Authors Journal compilation © 2012 FEBS.