During immune responses, B cells home to lymph nodes (LNs), where they encounter antigens. Homing starts with capture and L-selectin-dependent rolling on the activated endothelium of high endothelial venules (HEV). After recognition of chemokines presented on HEV, activation of B cell integrins occurs mediating firm arrest. Subsequently, B cells crawl to the spot of extravasation to enter the LN. Extravasation can be visualized and quantified in vivo by intravital microscopy (IVM) of the inguinal LN. Here, we describe an established protocol that permits detailed in vivo analysis of B cell recruitment to LN under sterile inflammatory conditions. We describe data acquisition, exportation, quantification, and statistical analysis using specialized software. IVM of LN is a powerful technique that can provide a better understanding of B cell migratory behavior during inflammation in vivo.Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.