Trastuzumab in combination with capecitabine or 5-fluorouracil and cisplatin is approved by the European Medicines Agency for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive (immunohistochemistry 3+ or immunohistochemistry 2+/fluorescence in situ hybridization-positive or immunohistochemistry 2+/silver in situ hybridization-positive) metastatic adenocarcinoma of the stomach or gastro-esophageal junction. Approvals are underway in other countries, with recent approvals granted in the United States and Japan. Experience and data from trastuzumab use in breast cancer have highlighted the importance of quality HER2 testing and scoring to ensure accurate identification of patients eligible for treatment. HER2 testing in gastric cancer differs from testing in breast cancer due to inherent differences in tumor biology; gastric cancer more frequently shows HER2 heterogeneity (focal staining) and incomplete membrane staining. Consequently, gastric cancer-specific HER2 testing protocols have been developed and standardized and it is imperative that these recommendations be adhered to. Given the predictive value of HER2 protein levels with response in the trastuzumab for GAstric cancer study (ToGA), immunohistochemistry should be the initial testing methodology and fluorescence in situ hybridization or silver in situ hybridization should be used to retest immunohistochemistry 2+ samples. Wherever possible, bright-field methodologies should be used as these are considered to be superior to fluorescent methodologies at identifying heterogeneous staining. Specific training is required before embarking on HER2 testing in gastric cancer, irrespective of the experience of HER2 testing in breast cancer. This paper provides the most up-to-date practical guidance on HER2 testing and scoring in patients with gastric and gastro-esophageal junction cancer, as agreed by a panel of expert pathologists with extensive experience of HER2 testing particularly reflecting the European Medicines Agency-approved indication. It is anticipated that these recommendations should ensure accurate and consistent HER2 testing, which will allow appropriate selection of patients eligible for treatment with trastuzumab.