Tumors develop through successive stages characterized by changes in gene expression and protein function. Gene expression profiling of pancreatic islet tumors in a mouse model of cancer revealed upregulation of cathepsin cysteine proteases. Cathepsin activity was assessed using chemical probes allowing biochemical and in vivo imaging, revealing increased activity associated with the angiogenic vasculature and invasive fronts of carcinomas, and differential expression in immune, endothelial, and cancer cells. A broad-spectrum cysteine cathepsin inhibitor was used to pharmacologically knock out cathepsin function at different stages of tumorigenesis, impairing angiogenic switching in progenitor lesions, as well as tumor growth, vascularity, and invasiveness. Cysteine cathepsins are also upregulated during HPV16-induced cervical carcinogenesis, further encouraging consideration of this protease family as a therapeutic target in human cancers.