Background: Neoadjuvant chemotherapy is administered prior to surgery for locally advanced tumors but does not confer any additional survival benefit. Measurement of Ki-67, a marker of cell proliferation, has been associated with response to therapy, but methods of measurement are controversial. There is no universal cut-point for association due to subjectivity in threshold for positivity and selection of the field of view. Here we propose that quantitative objective measurement for Ki-67 will provide a reproducible assay for likelihood of response to chemotherapy. Methods: A cohort of 115 consecutive (between 2002 and 2010) invasive breast cancer patients that received neoadjuvant therapy were included if pre-surgical biopsies were obtainable. Ki-67 expression was measured using quantitative immunofluorescence (AQUA) technology using the MIB-1 antibody. Images for each specimen were collected in 5 to 100 fields of view (FOV), and summary scores were obtained corresponding to the average and maximum of all the FOVs. Results: AQUA scoring was comparable to automated calculation of percent positive nuclei for prediction of response to chemotherapy (OR: 2.832 vs. 2.712). Both average and maximum AQUA scores showed Ki-67 expression was directly correlated to pathological complete response (pCR) (Ave p = 0.0002; Max p = 0.0011). Although examining the maximum field of view was more predictive of response to therapy (OR: 3.546 vs. 2.832), averaging all fields provided more sensitivity and specificity (AUC 0.769 vs. 0.732). Ki-67 average (p = 0.0025) and maximum (p = 0.0239) AQUA scores were also significant predictors of pCR in multivariable analysis with tumor size, nuclear grade, nodal status, ER status, and HER2 status considered. Conclusions: Measurement of Ki-67 expression by objective quantitative methods shows increased Ki-67 levels are an independent predictor of response to neoadjuvant chemotherapy. This assay is most sensitive and specific when the average Ki-67 expression from all fields of view is used.