The Ru-III-based prodrug AziRu efficiently binds to proteins, but the mechanism of its release is still disputed. Herein, in order to test the hypothesis of a reduction-mediated Ru release from proteins, a Raman-assisted crystallographic study on AziRu binding to a model protein (hen egg white lysozyme), in two different oxidation states, Ru-II and Ru-III, was carried out. Our results indicate Ru reduction, but the Ru release upon reduction is dependent on the reducing agent. To better understand this process, a pH-dependent, spectroelectrochemical surface-enhanced Raman scattering (SERS) study was performed also on AziRu-functionalized Au electrodes as a surrogate and simplest model system of Ru-II- and Ru-III -based drugs. This SERS study provided a pK(a) of 6.0 +/- 0.4 for aquated AziRu in the Ru-III state, which falls in the watershed range of pH values separating most cancer environments from their physiological counterparts. These experiments also indicate a dramatic shift of the redox potential E-0 by >600 mV of aquated AziRu toward more positive potentials upon acidification, suggesting a selective AziRu reduction in cancer lumen but not in healthy ones. It is expected that the nature of the ligands (e.g., pyridine vs imidazole, present in well-known Ru-III complex NAMI-A) will modulate the pK(a) and E-0, without affecting the underlying reaction mechanism.