Trastuzumab has widely been used to treat HER2-positive breast cancers. However, a large difference in clinical outcomes remains a key issue involving its treatment. Antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to be one of the modes of action for trastuzumab. The purpose of this study was to investigate the inter-individual differences in trastuzumab-mediated ADCC activity and develop a new assay to quantitative ADCC to predict the effecacy of trastuzmab. Using the peripheral blood mononuclear cells (PBMCs) of three healthy volunteers (HVs), we first examined ADCC. One of the HVs showed the highest ADCC against the HER2-positive BT-474 and MCF-7 cells, and we found in another independent experiment that the inter-individual differences among three subjects are constant. The stability of the inter-individual differences was also confirmed using PBMCs of an additional 8 HVs in three independent experiments. To search the molecules that correlate with ADCC activity, we adopted an new ex vivo gene expression assay. We examined the expression change of 14 candidate leucocyte genes in the 8 HVs after ex vivo exposure to heat-aggregated IgG1 for 4 h. The values of fold increase (FI) in expressions of three cytokines are significantly correlated with ADCC activity. Next, we evaluated prospectively whether FIs of these 14 genes are associated with a pathological complete response (pCR) in 18 patients who received trastuzumab-based neoadjuvant chemotherapy. Patients who achieved a pCR had a higher FI of four cytokines including prescribed two cytokines than those who did not.This is the first report of the consistent stability of inter-individual differences in trastuzumab-mediated ADCC activity in vitro and of a promising new assay for predicting the ADCC activity and the pathological response to trastuzumab-based neoadjuvant chemotherapy.