The generation of hybridoma cell lines by the fusion of splenocytes from immunized mice with immortal myeloma cells is a well-established method for the production of monoclonal antibodies. Although other methods have emerged as an effective alternative for the generation of monoclonal antibodies, the use of hybridoma technology remains a viable technique that is accessible to a wide number of laboratories that perform basic cell biological research. Hybridoma technology represents a relatively simple procedure at minimal cost for the continuous production of native whole immunoglobulins. This chapter will describe the materials and methodologies needed for the successful generation of monoclonal antibody (mAb)-producing hybridoma cell lines against target antigens.