Abstract Previous studies aimed at determining the affinity maturation pathways of monoclonal antibodies during an immunization have relied on comparing germ-line with mature forms of antibodies present in either transgenic B cell receptor mouse models or by cloning circulating human B cells present in peripheral blood (PB) of pre and post-immunized individuals. These approaches have been insightful, but they do not identify in confidence the true B cell clone(s) producing the antigen-specific antibodies found in the sera of immunized animals or humans. We recently developed a technology, NG-XMTTM, encompassing tandem mass spectrometry and next generation sequencing1,2, which for the first time enables the 1) identification of the anatomical location of the B cell clone(s) corresponding to the antigen-specific serum antibodies 2) investigation of clonal diversity of antigen-specific B cell clones residing in primary and secondary lymphoid organs and circulating in PB and 3) analysis of the evolving affinity mature serum antibodies, in two longitudinal immunization studies of rabbits and humans. These results not only advance our understanding of the affinity maturation process and the dynamics of memory B cells and plasma cell development that occurs during an immunization response, they also allow us to use this information to generate useful recombinant antibodies that can be utilized in passive immunization strategies. 1Nat Biotechnol. 2012,30:447; 2Nat Biotechnol. 2012,30:1039