Promising results have been reported in preclinical stroke target validation for pharmacological principles that disrupt the N-methyl-D-aspartate receptor–post-synaptic density protein-95–neuronal nitric oxide synthase complex. However, post-synaptic density protein-95 is also coupled to potentially neuroprotective mechanisms. As post-synaptic density protein-95 inhibitors may interfere with potentially neuroprotective mechanisms and sufficient validation has often been an issue in translating basic stroke research, we wanted to close that gap by comparing post-synaptic density protein-95 inhibitors with NOS1^−/− mice and a NOS inhibitor. We confirm the deleterious role of NOS1 in stroke both in vivo and in vitro, but find three pharmacological post-synaptic density protein-95 inhibitors to be therapeutically ineffective.