The nature of antibody-secreting cells in the rainbow trout is poorly defined. Here we describe a flow cytometric approach to help differentiate between four major trout B cell subsets present during terminal B cell differentiation: resting B cells, activated B cells, plasmablasts, and plasma cells. To aid in the identification of B cell subsets, the LPS-inducible transcription factor XbpI-S was used as a marker. An antibody specific to the stable form of inducible transcription factor X-box protein I (XbpI) was generated, which detects XbpI-S protein expression for species within the Oncorhyncus genus, including rainbow trout. Combinatorial expression patterns, or B cell signatures, were established using antibodies to XbpI-S, Pax5, and IgM in combination with a proliferation marker. We show that XbpI-S induction in trout splenic B cells increases throughout a 10-day in vitro LPS-induction period and that increased XbpI-S expression correlates with increased HCmu expression in the cell. PBLs displayed a lower level of XbpI-S induction during this incubation period, compared to spleen. We conclude that trout B cells follow a highly conserved B cell activation pathway, albeit slower than what has been observed in mammalian species. The use of XbpI-S as an activation marker for trout humoral immune activation promises to be useful for future in vivo studies, and can be applied to a broad range of teleost species.