Many forms of hematological malignancies as well as solid tumours can be treated using cellular immunotherapy, such as CAR-T, a treatment using T cells that express an antibody-based chimeric antigen receptor targeting tumor antigen. Due to the current challenges related to CAR-T cell therapy, as well as to investigate the potential of third-generation CARs, this study aimed to generate CAR-T cells by retroviral transduction and its sorting based on sGFP expression though Fluorescence-Activated Cell Sorting (FACS). The results obtained with flow cytometry analysis demonstrated approximately 52% of transduction efficiency. The confocal microscopy was able to detect sGFP signal, assessing the cellular distribution of sGFP-tagged CARs. The transduced cells were sorted using Fluorescence-Activated Cell Sorting (FACS) method. At the end of the process, one tube was obtained containing Jurkat cells highly expressing sGFP, corresponding to 46% of the original population.