Microbial infections can transit from their peripheral sites of entry and infection into the brain and central nervous system. Once there, they can wreak havoc on the control center for the entire body. The presence of culturable microbes in the brain and spinal cord is fairly easy to detect. Even unculturable microbes in the central nervous system can be detected by PCR and other assays. However, determining the mechanism by which these microbes cross the blood-brain barrier requires a collection of data. Herein, we delineate protocols for the in vivo, ex vivo, and in vitro analyses to determine the mechanism by which microbes cross the blood-brain barrier. These experimental techniques include flow cytometric analyses of brain extracts, histological analyses of brain sections, and in vitro Transwell coculture systems that mimic the blood-brain barrier.