Immunotherapy based on the adoptive transfer of genetically modified T lymphocytes for the expression of engineered receptors has provided impressive results for hematological neoplasms. This is the case of T cells modified to express chimeric antigen receptors (CAR). However, for solid neoplasms, which represent most of the tumors, several limitations still need to be overcome for the development of more effective and safe therapies. In the case of CAR-T cells, one of the limitations in their application for solid tumors is their restricted ability to recognize only surface antigens in their native conformation. Nonetheless, engineered T lymphocytes expressing artificial T cell receptor (TCR) with enhanced affinity for a specific tumor antigen, referred as TCR-T cells, have provided encouraging results against solid tumors. The artificial TCR, as well as the endogenous TCR, is able to recognize intracellular proteins presented by the major histocompatibility complex (MHC), enabling a diversity of options for therapeutic targets, since most tumor-associated antigens are intracellular. The NY-ESO-1 is a potent target in this therapeutic approach and the most used in TCR-T cells studies. This intracellular protein has restricted expression in healthy tissues, found in the placenta and in immune privileged tissues, and has strong and homogeneous expression in different types of tumors. Therefore, the main goal of this study was the generation of TCR-T cells targeting the peptide:HLA complex formed by NY-ESO-1:HLA-A*02 using a new genetic construct of artificial TCR. For this, human T lymphocytes were transduced (MOI 5) with a lentiviral vector for the expression of α and β chains of the engineered TCR. The transduction efficiency as 11% as determined by labeling with NY-ESO-1:HLA-A*02 specific tetramers. Next, TCR-T cells were co-cultured with SW982 sarcoma cells (NY-ESO-1+/HLA-A*02+) or HCT116 colon carcinoma cells (NY-ESO-1−/HLA-A*02+) to evaluate their in vitro antitumor capacity. TCR-T cells were able to eliminate approximately 30% of the SW982 cells after 48h of co-culture in a 1:1 ratio (effector:target cells). Additionally, these cells had no cytotoxic capacity against HCT116 cells. Together, these data demonstrated the in vitro antitumor potential and the specificity of TCR-T cells anti-NY-ESO-1:HLA-A*02 to the target antigen, validating these new genetic construct. Thus, these results are encouraging and promising in view of the development of a new advanced cell therapy for solid neoplasms.