Background: Lymphocytes proliferate considerably following appropriate stimulation in vitro. Autologous T cells are obtained from whole blood or tissue sites in relatively limited amounts. We need a method to expand these cells efficiently, study their functions and manipulate them to create appropriate cells for transferring to the patient with infection and cancer. Objectives: The aim of this study is to determine proliferation ability of two different stimulators on CD4+ lymphocytes. Methods: Lymphocytes were isolated from blood samples of healthy donors after removing adherent cells (monocytes).The efficacy of MACSiBead™ coated with anti-CD2, anti-CD3, anti-CD28 (anti-CD2/CD3/CD28) was compared with Phytohaemagglutinin A (PHA) on CD4+ lymphocytes proliferation using carboxyfluorescein diacetate succinimidyl ester (CFSE) in cell culture media. The percentage of proliferating cells was analyzed using flow cytometry. Results: Both stimulators induced extensive proliferation of CD4+ lymphocytes but proliferation ability of PHA was higher compared to stimulation by anti-CD2/CD3/CD28 MACSiBead™. The proliferation rate of cells stimulated by PHA was 93.8% ± 3.37% whereas it was 85.2% ± 4.7% in cells stimulated by anti-CD2/CD3/CD28 MACSiBead™. Conclusions: Our results show that MACSiBead™ along with PHA can be used to obtain a large number of expanded CD4+ lymphocytes.