Objective Although several methods have been reported and used for in vitro T cell amplification, there are no consistent reports on the optimal stimulation conditions and the characterization of these stimulated T cells. The current study aimed to determine the optimal conditions for efficient T cell amplification by two commonly used methods involving CD3/CD28 antibody and phytohemagglutinin (PHA), respectively. Results Orthogonal design and CCK8 assay showed that 5 mu g/mL CD3, 5 mu g/mL CD28, and 100 ng/mL IL2 for the first method and 50 mu g/mL PHA for the second method was optimal for T cell stimulation. Flow cytometry demonstrated that the percentage of CD8(+) in the stimulated groups significantly increased, while the percentage of CD4(+)/CD8(+) was significantly decreased compared with the unstimulated group. The percentage of CD4(+) showed no significant difference among the three groups. Notably, there was no significant difference between the two stimulated groups. In addition, the percentage of apoptotic cells was significantly increased in the stimulated groups compared with the unstimulated group, but showed no remarkable difference between the PHA and CD3/CD28 stimulation groups. Glycolysis analysis showed that the glycolytic capacity and glycolytic reserve were both significantly increased in the PHA and CD3/CD28 groups compared with the unstimulated group, with no significant difference noted between the stimulated groups. Conclusions Although both stimulation methods showed similar efficacies, we suggest the PHA method might be better considering its easy application and cost-effective nature.