Recombinant D-amino acid oxidase (DAAO) is extensively employed for controlled generation of intracellular HO. This chemogenetic tool offers unique opportunities for dissecting the contribution of oxidative stress in the occurrence of aging-related degenerative disorders. However, the employment of this tool to induce oxidative stress in the brain in vivo has not yet been tested. Here, we combine viral vector-mediated expression of DAAO and HyPer7, a genetically encoded fluorescent biosensor for HO, in hippocampal neurons with real-time in vivo fiber photometry to examine chemogenetic production of HO upon delivery of D-norvaline via intraperitoneal injections or drinking water. Observed changes in the HyPer7 ratiometric signal in response to D-norvaline delivery through both routes confirmed controlled generation of intraneuronal HOin vivo. Raman microspectroscopy revealed a decrease of electron transport chain loading with electrons upon in vivo chemogenetic generation of intraneuronal HO. In sum, our findings enable us to suggest that the DAAO-based chemogenetic tool combined with delivery of D-norvaline via either intraperitoneal injections or drinking water is suitable for induction of chronic oxidative stress in the brain in vivo.