Previous studies have highlighted the importance of an appropriate human epidermal growth factor receptor 2 (HER2) evaluation for the proper identification of patients eligible for treatment with anti-HER2 targeted therapies. Today, the relationship remains unclear between the level of HER2 amplification and the outcome of HER2-positive gastric cancer treated with first-line chemotherapy with trastuzumab. The aim of this study was to determine whether the level of HER2 gene amplification determined by the HER2/CEP17 ratio and HER2 gene copy number could significantly predict some benefit in overall survival and response to therapy in advanced gastric cancer treated with trastuzumab-based chemotherapy. Ninety patients with metastatic gastric cancer treated with first-line trastuzumab-based chemotherapy were studied. The optimal cutoff values for HER2/CEP17 ratio and HER2 gene copy number (GCN) for discriminating positive results in terms of response and prolonged survival were determined using receiver operating characteristic curves analyses. In this study, a median HER2/CEP17 ratio of 6.11 (95% CI, 2.27 to 21.90) and a median HER2 gene copy number of 11.90 (95% CI, 3.30 to 43.80) were found. A mean HER2/CEP17 ratio of 4.7 was identified as the optimal cutoff value discriminating sensitive and refractory patients (P = .005). Similarly, the optimal cutoff for predicting survival longer than 12 months was 4.45 (P = .005), and for survival longer than 16 months was 5.15 (P = .004). For HER2 GCN, the optimal cutoff values were 9.4, 10.0, and 9.5, respectively (P = .02). The level of HER2 gene amplification significantly predicts sensitivity to therapy and overall survival in advanced gastric cancer treated with trastuzumab-based chemotherapy.