Using an intraoperative margin assessment technique during breast-conserving surgery (BCS) helps surgeons to decrease the risk of positive margin occurrence. Diffuse reflectance spectroscopy (DRS) has the potential to discriminate healthy breast tissue from cancerous tissue. We investigated the performance of an electrosurgical knife integrated with a DRS on porcine muscle and adipose tissue. Characterization of the formed debris on the optical fibers after electrosurgery revealed that the contamination is mostly burned tissue. Even with contaminated optical fibers, both tissues could still be discriminated with DRS based on fat/water ratio. Therefore, an electrosurgical knife integrated with DRS may be a promising technology to provide the surgeon with real-time guidance during BCS.