CRISPR/Cas9 is a gene editing technology that can correct and cut specific DNA sequences. Using CRISPR/Cas9, T Cell Receptor (TCR) can be edited to increases the specificity of the immune response. However, when using CRISPR/Cas9, there are difficulties of necessity for activation and transformation efficiency. In this study, we established a serum-free condition for gene editing of human primary T cells. We also verified that knockout (KO) is possible not only surface molecular but also intracellular kinase. Finally, we also showed that a mixture of guide RNAs (gRNAs) targeting a single gene or multiple ribonucleoproteins (RNPs) for plural gene locus resulted in highly efficient deletion of TCR genes. In conclusion, this study suggests the importance of activation and the feasibility of utilizing multiple gRNAs or RNPs to increase the efficiency of KO of primary T cells using CRISPR-Cas9 in serum-free conditions.