Complete tumor removal during surgery has a great impact on patient survival. To that end, the surgeon should detect the tumor, remove it and validate that there are no residual cancer cells left behind. Residual cells at the incision margin of the tissue removed during surgery are associated with tumor recurrence and poor prognosis for the patient1. Therefore, there is a need for diagnostic tools that will differentiate between the tumor and its normal surroundings. We designed Turn-ON probes that will be activated by Cathepsin B (CTB), a cysteine protease that is highly expressed in multiple tumors. To date, numerous low molecular weight CTB-sensitive substrates have been reported, however, most of them suffer from rapid clearance and reduced signal shortly after administration. We present herein the synthesis and characterization of three novel polymeric Turn-ON probes that are activated at the tumor site by CTB. Utilizing orthotopic breast cancer and melanoma models, we studied the kinetics of the polymeric nano-probes, and showed an improved tumor-to-background ratio upon activation by CTB compared to currently-available systems. The signal obtained from the tumor was stable and delineated the tumor boundaries during the whole surgical procedure, enabling accurate resection. Our findings show that the control group of tumor-bearing mice, that underwent standard surgery without fluorescence real-time guidance, survived less time and suffered from tumor recurrence earlier than the group that underwent image-guided surgery (IGS) using our Turn-ON probes. Our "smart" polymeric probes can potentially assist surgeons’ decision in real-time during surgery regarding the tumor margins needed to be removed, leading to improved patient outcome.