Simple SummaryBreast-conserving surgery (BCS) is an oncological procedure that allows for the excision of breast cancer with a clear margin of healthy tissue whilst optimising the cosmetic appearance. However, BCS is associated with up to a 19% re-excision rate due to incomplete excision (“positive margins”) in the United Kingdom. Optical spectroscopy and the optical imaging of BCS specimens could be a potential intraoperative margin assessment tool to help reduce re-excision rates. Hyperspectral sensing is based on the premise that light illuminating biological tissues undergoes several processes that reflect the composition of tissue, thus helping to differentiate between normal and malignant tissues. This review assesses the current literature on the use of hyperspectral sensing in breast cancer. We divide the techniques into either point-based (spectroscopy) or whole field-of-view (imaging) methods. A comparison is made of the effectiveness of these modalities in discriminating between normal and malignant tissue, and we reflect on the usability of these modalities in the intraoperative setting.AbstractUp to 19% of patients require re-excision surgery due to positive margins in breast-conserving surgery (BCS). Intraoperative margin assessment tools (IMAs) that incorporate tissue optical measurements could help reduce re-excision rates. This review focuses on methods that use and assess spectrally resolved diffusely reflected light for breast cancer detection in the intraoperative setting. Following PROSPERO registration (CRD42022356216), an electronic search was performed. The modalities searched for were diffuse reflectance spectroscopy (DRS), multispectral imaging (MSI), hyperspectral imaging (HSI), and spatial frequency domain imaging (SFDI). The inclusion criteria encompassed studies of human in vivo or ex vivo breast tissues, which presented data on accuracy. The exclusion criteria were contrast use, frozen samples, and other imaging adjuncts. 19 studies were selected following PRISMA guidelines. Studies were divided into point-based (spectroscopy) or whole field-of-view (imaging) techniques. A fixed-or random-effects model analysis generated pooled sensitivity/specificity for the different modalities, following heterogeneity calculations using the Q statistic. Overall, imaging-based techniques had better pooled sensitivity/specificity (0.90 (CI 0.76–1.03)/0.92 (CI 0.78–1.06)) compared with probe-based techniques (0.84 (CI 0.78–0.89)/0.85 (CI 0.79–0.91)). The use of spectrally resolved diffusely reflected light is a rapid, non-contact technique that confers accuracy in discriminating between normal and malignant breast tissue, and it constitutes a potential IMA tool.