Optogenetics and functional magnetic resonance imaging (fMRI) are two tools that have become indispensable for neuroscientists, though they have traditionally been used to ask distinct questions regarding neurophysiology. Namely, optogenetics is used to manipulate neuronal activity by activating or inhibiting specific brain regions and/or neuronal subtypes, while fMRI detects and quantifies brain-wide activity and dynamics under different conditions. However, when combined, optogenetics and fMRI (opto-fMRI) can be a powerful technique for understanding the brain-wide effects of stimulating or inhibiting a specific brain region/neuronal subtype. Opto-fMRI has mainly been applied under “baseline” conditions but holds promise for understanding pathological processes in neurological disorders. Here, we describe the general setup for opto-fMRI experiments and their potential for translational relevance, using our own experiments as a proof-of-concept. We stress the importance of carefully considered control groups and proper confirmation of optogenetic function in employing opto-fMRI. Moreover, beyond traditional measures of static functional connectivity, additional analyses of dynamic functional connectivity could shed light on currently unexplored disease processes and potential therapeutic avenues. Finally, although we highlight the application of opto-fMRI using the locus coeruleus–norepinephrine system in an Alzheimer’s disease model rat as an example, this technique is broadly applicable for the study of other neurological and neuropsychiatric diseases.