Background: Cutaneous leishmaniasis is a zoonotic disease transmitted between rodents and canine, mainly induced by phelebotomus sand flies. In Iran, the incidence of this protozoan disease has doubled over the last decade. Regard to its high incidence, in the present study the new antigen preparation was evaluated following challenging with live Leishmania major promastigts. Methods: One hundred and twenty male and female Balb/c mice were randomly divided into four groups as LT, LB, LBT and control groups. Group LT received 100-200 μg /0.1 ml of the crude cocktail antigen preparation plus alcoholic extract of Teucrium polium as adjuvant, LB received 100-200 μg /0.1 ml of the crude cocktail antigen preparation plus BCG as adjuvant, LBT received 100-200 μg /0.1 ml of this antigen preparation plus alcoholic extract of Teucrium polium and BCG as adjuvants and control group. Groups LT, LB and LBT were injected subcutaneously with the antigen preparation and they were boosted using the same doses with two weeks intervals later. A week after the last booster, all animals was challenged with live Leishmania major promastigotes. The animals survived, were euthanized, their spleen was removed, and 4-5 microns paraffin sections were prepared and stained with Harris Hematoxylin and Eosine method. The expansion rates of the spleen white pulp size were evaluated using a light microscope with eye-piece graticule. Results & Conclusion: Statistically, Higher expansion rate of the spleen white pulp size were recorded in the LBT group. The antigen preparation supplemented with adjuvants had positive effects on spleen white pulp expansion following live Leishmania major challenging, nevertheless, based on previous experiment, 100-200 μg of LT group could induces satisfactory DTH response which favors leishmania organisms eradication, and higher survival rate after challenging with the live Leishmania major organisms in spite of induction of lower spleen white pulp size expansion.