Epithelial cancers are often infiltrated with prognostically relevant lymphocytes, including T and B lymphocytes. Isolation, expansion, and immortalization of lymphocytes from epithelial tumors are immensely important in basic tumor immunology research and clinical translation. Here, we outline in detail the process of separating viable B lymphocytes from surgically removed epithelial tumors and their immortalization and cryopreservation methods. Overall, this state-of-the-art procedure aids in bulk and specific antigen-reactive antibody production from tumor-infiltrating B cells. Also, sequence information can be deduced by cutting-edge sequencing methods from these immortalized pools of B lymphocytes.