Calcium (Ca2+) flux acts as a central signaling pathway in B cells, and its alterations are associated with autoimmune dysregulation and B-cell malignancies. We standardized a flow-cytometry-based method using various stimuli to investigate the Ca2+flux characteristics of circulating human B lymphocytes from healthy individuals. We found that different activating agents trigger distinct Ca2+flux responses and that B-cell subsets show specific developmental-stage dependent Ca2+flux response patterns. Naive B cells responded with a more substantial Ca2+flux to B cell receptor (BCR) stimulation than memory B cells. Non-switched memory cells responded to anti-IgD stimulation with a naive-like Ca2+flux pattern, whereas their anti-IgM response was memory-like. Peripheral antibody-secreting cells retained their IgG responsivity but showed reduced Ca2+responses upon activation, indicating their loss of dependence on Ca2+signaling. Ca2+flux is a relevant functional test for B cells, and its alterations could provide insight into pathological B-cell activation development.