Purpose In the I-SPY 2 TRIAL (Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis 2), the pan-erythroblastic oncogene B inhibitor neratinib was available to all hormone receptor (HR)/human epidermal growth factor receptor 2 (HER2) subtypes and graduated in the HR-negative/HER2-positive signature. We hypothesized that neratinib response may be predicted by baseline HER2 epidermal growth factor receptor (EGFR) signaling activation/phosphorylation levels independent of total levels of HER2 or EGFR proteins. Materials and Methods Complete experimental and response data were available for between 130 and 193 patients. In qualifying analyses, which used logistic regression and treatment interaction analysis, 18 protein/phosphoprotein, 10 mRNA, and 12 DNA biomarkers that related to HER family signaling were evaluated. Exploratory analyses used Wilcoxon rank sum and t tests without multiple comparison correction. Results HER pathway DNA biomarkers were either low prevalence or nonpredictive. In expression biomarker analysis, only one gene (STMN1) was specifically associated with response to neratinib in the HER2-negative subset. In qualifying protein/phosphoprotein analyses that used reverse phase protein microarrays, six HER family markers were associated with neratinib response. After analysis was adjusted for HR/HER2 status, EGFR Y1173 (pEGFR) showed a significant biomarker-by-treatment interaction (P =.049). Exploratory analysis of HER family signaling in patients with triple-negative (TN) disease found that activation of EGFR Y1173 (P =.005) and HER2 Y1248 (pHER2) (P =.019) were positively associated with pathologic complete response. Exploratory analysis in this pEGFR/pHER2-activated TN subgroup identified elevated levels of estrogen receptor α (P <.006) in these patients. Conclusion Activation of HER family phosphoproteins associates with response to neratinib, but only EGFR Y1173 and STMN1 appear to add value to the graduating signature. Activation of HER2 and EGFR in TN tumors may identify patients whose diseases respond to neratinib and implies that there is a subset of patients with TN disease who paradoxically exhibit HER family signaling activation and may achieve clinical benefit with neratinib; this concept must be validated in future studies.