Abstract Background The human epidermal growth factor receptor-2 (HER-2) expression level is a critical element for determining the prognosis and management of breast cancer. HER-2 targeted therapy in breast cancer depends on the reliable assessment of HER-2 expression status but current standard methods are lacking a rigorous quantitative assay. To address this challenge, we developed an assessment of HER-2 expression method by well-based reverse phase protein array (RPPA). Results Well-based RPPA is based on a robust protein isolation methodology paired with a novel electrochemiluminescence detection system. HER-2 value of well-based RPPA significantly correlated with dot blotting results (R 2  = 0.939). By well-based RPPA, we successfully detected HER-2 expression in 76 human breast formalin-fixed paraffin-embedded tissue samples. We observed 93.4% (71/76) concordance between well-based RPPA and current HER-2 immunohistochemical assessment guideline. When the cutoff level of HER-2 value was set to 0.689 (HER-2/GAPDH) on the basis of receiver-operating characteristic curve, the area under the curve was 0.975 (95% CI, 0.941-1.000). Sensitivity and specificity of well-based RPPA was 92.1% and 94.7%, respectively. Conclusions HER-2 value by well-based RPPA was correlated with the current HER-2 status guideline, suggesting that this normalized HER-2 assessment may offer advantages over unnormalized current immunohistochemical assessment methods.