The study aims to assess site assessment of the performance of ¹⁸F-PBR-111 as a neuroinflammation marker in the cuprizone mouse model of multiple sclerosis (MS). ¹⁸F-PBR-111 PET imaging has not been well evaluated in multiple sclerosis applications both in preclinical and clinical research. This study will help establish the potential utility of ¹⁸F-PBR-111 PET in preclinical MS research and future animal and future human applications. ¹⁸F-PBR-111 PET/CT was conducted at 3.5 weeks (<i>n</i> = 7) and 5.0 weeks (<i>n</i> = 7) after cuprizone treatment or sham control (<i>n</i> = 3) in the mouse model. A subgroup of mice underwent autoradiography with cryosectioned brain tissue. T2 weighted MRI was performed to obtain the brain structural data of each mouse. ¹⁸F-PBR-111 uptake was assessed in multiple brain regions with PET and autoradiography images. The correlation between autoradiography and immunofluorescence staining of neuroinflammation (F4/80 and CD11b) was measured. Compared to control mice, significant ¹⁸F-PBR-111 uptake in the corpus callosum (<i>p</i> < 0.001), striatum (caudate and internal capsule, <i>p</i> < 0.001), and hippocampus (<i>p</i> < 0.05) was identified with PET images at both 3.5 weeks and 5.0 weeks, and validated with autoradiography. No significant uptake differences were detected between 3.5 weeks and 5.0 weeks assessing these regions as a whole, although there was a trend of increased uptake at 5.0 weeks compared to 3.5 weeks in the CC. High ¹⁸F-PBR-111 uptake regions correlated with microglial/macrophage locations by immunofluorescence staining with F4/80 and CD11b antibodies. ¹⁸F-PBR-111 uptake in anatomic locations correlated with activated microglia at histology in the cuprizone mouse model of MS suggests that ¹⁸F-PBR-111 has potential for in vivo evaluation of therapy response and potential for use in MS patients and animal studies.