Protocols for efficient capture of antigen-specific B cells (ASBCs) are useful for understanding pathogen-specific B-cell responses during natural infection or vaccination. Fluorescently labeled tetramerized probes are classically used to capture ASBCs, but many occlude valuable epitopes available for B-cell receptor binding. Here, we describe a bead assay to confirm ASBC receptor accessibility on probes and a sequential staining process to capture HIV gp140-specific B cells from human peripheral blood mononuclear cells. For complete details on the use and execution of this protocol, please refer to Townsley et al. (2021).Keywords: Flow Cytometry/Mass Cytometry; Immunology; Molecular/Chemical Probes.