The successful use of Chimeric Antigen Receptor (CAR) for hematological cancer treatment has influenced the direction taken in translational research towards an increasing focus on personalized targeted immunotherapy. Thus, a growing number of labs worldwide are now interested in testing their old antibody collections in this format to broaden the spectrum of utility, and improve safety and efficacy. We herein present a straightforward protocol for the identification of an antibody from a hybridoma and the design of the single chain fragment that will be placed on the extracellular part of the CAR construct. We further show how to test the expression and the activity of the construct in primary T cells. We illustrate our demonstration with two new CARs targeted against the B cell receptor (BCR), more precisely the light chains κ and λ, which represent potential alternatives to the CD19 CAR used in the treatment of B-cell malignancies.