Proteases are enzymes that primarily function by degrading protein substrates. They often play pathogenic roles in common human diseases such as cancer, asthma, arthritis, atherosclerosis and infection by pathogens. Therefore tools that can be used to dynamically monitor their activity in vivo can be used as diagnostic agents and as imaging contrast agents for intraoperative image guidance. In this presentation, I will describe the design and synthesis of a series of near infrared fluorogenic probes that exploit a latent cationic lysosomotropic effect (LLE) to promote cellular retention upon protease activation. I will also describe a new dual optical/PET covalent cathepsin probe that can be used for non-invasive imaging applications. The fluorogenic LLE probes show tumor-specific retention, fast activation kinetics, and rapid systemic distribution. Furthermore they are suitable for detection of diverse cancer types including breast, colon and lung tumors. Most importantly, the agents are compatible with the existing, FDA approved, da Vinci® surgical system for fluorescence guided tumor resection. Therefore, our data suggest that small molecule fluorescent reporters can be used with existing clinical instrumentation to detect tumors and potentially other types of inflammatory lesions for both early diagnosis and to guide surgical decision making in real time. I will also present our first-in-human studies using the cathepsin targeted optical/PET probe to image lung fibrosis in patients with idiopathic pulmonary fibrosis (IPF).