Introduction: Epstein-Barr virus encoded RNA (EBER) in situ hybridization (ISH) is a method for the identification of Epstein-Barr virus (EBV) infection in tissue sections. In our experience, the currently available automated EBER ISH staining protocol using the Bond-Max immunostainer is not suitable for xenograft tissue sample due to high background staining which impairs the interpretation of the staining. Methods: Here we describe a modified automated EBER ISH staining protocol for xenografts which involves the substitution of reagents for detecting the EBER probe. Results: This modified protocol significantly improved the quality and speed of the staining. Conclusion: The method described here will be useful for EBER ISH staining of xenograft tissue samples.