PURPOSE: This study evaluated pathological response to neoadjuvant chemotherapy using quantitative ultrasound (QUS) and diffuse optical spectroscopy imaging (DOSI) biomarkers in locally advanced breast cancer (LABC). MATERIALS AND METHODS: The institution's ethics review board approved this study. Subjects (n = 22) gave written informed consent prior to participating. US and DOSI data were acquired, relative to the start of neoadjuvant chemotherapy, at weeks 0, 1, 4, 8 and preoperatively. QUS parameters including the mid-band fit (MBF), 0-MHz intercept (SI), and the spectral slope (SS) were determined from tumor ultrasound data using spectral analysis. In the same patients, DOSI was used to measure parameters relating to tumor hemoglobin and composition. Discriminant analysis and receiver-operating characteristic (ROC) analysis was used to classify clinical and pathological response during treatment and to estimate the area under the curve (AUC). Additionally, multivariate analysis was carried out for pairwise QUS/DOSI parameter combinations using a logistic regression model. RESULTS: Individual QUS and DOSI parameters, including the (SI), oxy-hemoglobin (HbO2), and total hemoglobin (HbT) were significant markers for response after one week of treatment (p < 0.01). Multivariate (pairwise) combinations increased the sensitivity, specificity and AUC at this time; the SI + HbO2 showed a sensitivity/specificity of 100%, and an AUC of 1.0. CONCLUSIONS: QUS and DOSI demonstrated potential as coincident markers for treatment response and may potentially facilitate response-guided therapies. Multivariate QUS and DOSI parameters increased the sensitivity and specificity of classifying LABC patients as early as one week after treatment.