Proper blood-brain barrier (BBB) function is essential to maintain homeostasis, keeping the central nervous system healthy. Disruption of the BBB has been described in nearly all neurological disease and injury contexts. Given the various physical and metabolic roles of the BBB and gliovascular unit (GVU), dysfunction should be assessed across multiple levels to pinpoint the exact nature of the impairment. Here, we describe imaging techniques for evaluating various aspects of BBB and GVU integrity and function, tailored for use in mouse models, and accompanied by protocols for quantitative image analysis. These imaging techniques complement classical dye tracer experiments and reveal underlying molecular causes of BBB dysfunction.