Complete removal of malignant tissue during primary breast cancer resection is a critical prognostic indicator of local recurrence and overall patient survival, making intraoperative tumor margin assessment essential. Positive margin status following breast-conserving surgery (BCS) is a common difficulty reported in 20-60% of patients, with re-excision rates >55%. Re-excision increases the risk of morbidity and delays the use of adjuvant therapy, thus significant efforts are underway to develop successful intraoperative margin assessment strategies to eliminate repeat surgery. One novel strategy uses topical application of dual probe staining and a fluorescence imaging strategy termed dual probe difference specimen imaging (DDSI) where a receptor-targeted fluorescent probe and an untargeted, spectrally-distinct fluorescent probe are topically applied to the fresh resected specimen. While conceptually simple, resected specimen staining is dominated by non-specific uptake of fluorescent probes in normal tissue, requiring the use of a dual probe strategy for accuracy. DDSI permits fluorescence images from both the targeted and untargeted probes to be used to calculate a normalized difference image, facilitating quantitative identification of targeted probe tumor distribution in the specimen. While previous reports suggested this approach is a promising new tool for surgical guidance, advancing the approach into the clinic requires methodical protocol optimization and validation. Current work is focused on development of targeted and untargeted small molecule affinity tags, facilitating access to intracellular breast cancer biomarkers and quantitative assessment of DDSI signal in the context of varied biomarker expression level.