The selective and temporal control of protein activity in living cells provides a powerful tool to manipulate cellular function and to develop pro-protein therapeutics (PPT) for targeted therapy. In this work, we reported a facile but general chemical approach to design PPT by modulating protein activity in response to endogenous enzyme of disease cells, and its potential for targeted cancer therapy. We demonstrated that the chemical modification of a protein with quinone propionic acid (QPN), a ligand that could be reduced by tumor-cell-specific NAD(P)H dehydrogenase [quinone] 1 (NQO1), was reversible in the presence of NQO1. Importantly, the QPN-modified cytochrome c (Cyt c-QPN) and ribonuclease A (RNase A-QPN) showed NQO1-regulated protein activity in a highly selective manner. Furthermore, the intracellular delivery of RNase A-QPN using a novel type of lipid-based nanoparticles, and subsequent protein activation by cellular NQO1, selectively inhibit cancer cell growth in vitro and effectively suppress tumor growth in vivo. We believe that our approach increases the number of potentially useful chemical tools for reversibly controlling the structure and function of protein using a disease-cell-specific enzyme, opening opportunities in the study of dynamic biological processes and developing precise protein therapeutics.