Objective To study the optimum determination conditions for lymphocytic proliferation by CCK-8 method in mice.Methods To study the different influence factors of spleen cell proliferation experiment stimulated by mitogen concanavalin A (ConA) or lipopolysaccharide (LPS),including cell preparation method,lymphocytic density,FBS and stimulating agent concentration in culture medium,and stimulating immediately or 24 h after preparing cell,with cross design or two factor completely randomized design.Results Spleen lymphocytic proliferation rate of preparation method by light suppression was higher than that of the light grind.The appropriate concentration of spleen cells was 5 × 106/mL.The proliferation rate has no significant difference after being stimulated for 48 or 72 h by ConA (2,5,or 1 0 μg/mL) or LPS (10,20,or 50 μg/mL) under 10％,15％,or 20％ FBS concentration in culture medium.The proliferation rate of stimulating immediately after preparing cell was higher than that of 24 h after preparing cell.Conclusion The optimum conditions of Balb/C mouse spleen cell proliferation assay stimulated by ConA and LPS are as follows:preparation of spleen cells with light pressure,spleen cell concentration of 5 × 106/mL,direct stimulation with 2-10 μg/mL ConA or 10-50 μg/mL LPS in the day of preparation.