It has been a long-term goal to develop cancer-imaging techniques that have sufficient specificity and sensitivity, since early detection and complete resection are an important prognosticator for cancer treatment. Since fluorescence-guided diagnosis is one of the most powerful techniques for real-time in situ cancer detection, we have focused on developing a cancer-imaging method with activatable fluorescent probes targeted to enzymes that are overexpressed in cancer. These include probes for cancer-associated aminopeptidases, carboxypeptidases, and glycosidases, which allowed us to visualize certain types of cancers in the resected clinical specimens from the patients. In this presentation, I would like to introduce the molecular design, our recent effort for screening the candidate probes from our probe library, and its application for clinical specimens. We hope that fluorescence-guided diagnosis by our activatable probes can be used as an imaging guidance during surgical procedures to help surgeons for efficient tumor inspection.