This chapter introduces a protocol for immunophenotyping B-cell subsets using flow cytometry, a vital technique in both immunology research and clinical practice. Flow cytometry offers detailed insights into B-cell diversity and function, aiding in understanding B-cell development and guiding therapeutic decisions. This chapter delves into critical aspects such as antibody panels, sample preparation, and data analysis, all of which are essential for effectively analyzing B-cell subpopulations in diagnosing conditions like common variable immunodeficiency and monitoring therapeutic outcomes after B-cell depletion. The described protocol ensures accurate quantitative and qualitative determination of total B cells and subpopulations, providing a valuable resource for researchers and clinicians alike.