AbstractBackground and ObjectiveMost women with early stage breast cancer have the option of breast conserving therapy, which involves a partial mastectomy for removal of the primary tumor, usually followed by radiotherapy. The presence of tumor at or near the margin is strongly correlated with the risk of local tumor recurrence, so there is a need for a non‐invasive, real‐time tool to evaluate margin status. This study examined the use of autofluorescence and diffuse reflectance spectroscopy and spectral imaging to evaluate margin status intraoperatively.Materials and MethodsSpectral measurements were taken from the surface of the tissue mass immediately following removal during partial mastectomies and/or from tissues immediately after sectioning by surgical pathology. A total of 145 normal spectra were obtained from 28 patients, and 34 tumor spectra were obtained from 12 patients.ResultsAfter correlation with histopathology, a multivariate statistical algorithm classified the spectra as normal (negative margins) or tumor (positive margins) with 85% sensitivity and 96% specificity. A separate algorithm achieved 100% classification between neo‐adjuvant chemotherapy‐treated tissues and non‐treated tissues. Fluorescence and reflectance‐based spectral images were able to demarcate a calcified lesion on the surface of a resected specimen as well.ConclusionFluorescence and reflectance spectroscopy could be a valuable tool for examining the superficial margin status of excised breast tumor specimens, particularly in the form of spectral imaging to examine entire margins in a single acquisition. Lasers Surg. Med. 42:15–23, 2010. © 2010 Wiley‐Liss, Inc.