Toxoplasma gondii is an obligate intracellular, protozoan pathogen of rodents and humans. T. gondii's ability to grow within cells and evade cell-autonomous immunity depends on the integrity of the parasitophorous vacuole (PV). Interferon-inducible guanylate binding proteins (GBPs) are central mediators of T. gondii clearance, however, the precise mechanism linking GBP recruitment to the PV and T. gondii restriction is not clear. This knowledge gap is linked to heterogenous GBP-targeting across a population of vacuoles and the lack of tools to selectively purify the intact PV. To identify mediators of parasite clearance associated with GBP2-positive vacuoles, we employed a novel protein discovery tool automated spatially targeted optical micro proteomics (autoSTOMP). This approach identified inducible nitric oxide synthetase (iNOS) enriched at levels similar to the GBPs in infected bone marrow-derived myeloid cells. iNOS expression on myeloid cells was necessary for mice to control T. gondii growth in vivo and survive acute infection. T. gondii infection of IFNγ-primed macrophage was sufficient to robustly induce iNOS expression. iNOS restricted T. gondii infection through nitric oxide synthesis rather than arginine depletion, leading to robust and selective nitration of the PV. Optimal parasite restriction by iNOS and vacuole nitration depended on the chromosome 3 GBPs. Notably, GBP2 recruitment and ruffling of the PV membrane occurred in iNOS knockouts, however, these vacuoles contained dividing parasites. iNOS activity was necessary for the collapse of the intravacuolar network of nanotubular membranes which connects parasites to each other and the host cytosol. Based on these data we conclude reactive nitrogen species generated by iNOS cooperate with the chromosome 3 GBPs to target distinct biology of the PV that are necessary for optimal parasite clearance in murine myeloid cells.