Staphylococcus aureus (S. aureus) was the most prevalent bacteria recorded from clinical and subclinical mastitis. The main objective of the present work is to prepare and evaluate the effectiveness of locally prepared inactivated S. aureus vaccine using different adjuvants Montanide ISA-206, Montanide ISA-70 and alum hydroxide gel for detection of cellular and humeral immune responses. Seventy dairy heifers in the last trimester of gestation divided into (5) groups. Twenty pregnant cows in three groups (G1, G2, and G3) were inoculated intramuscularly with the prepared vaccines four weeks prior to calving then a booster dose at two weeks pre-expected calving. The animals in remaining groups (G4 and G5) were considered as control and safety test respectively. Serum samples from vaccinated and non-vaccinated cows were collected at the 1st and 2nd immunization then at 0, 1, 2, 3 months and (7, 10, 14) days post calving and vaccination and evaluated immunologically using ELISA and Lymphocyte transformation assay, respectively. The results revealed that a progressive increase of immune response in the vaccinated groups (G1 and G2) than that of the third and control groups. These results could be indicated the safety and effectiveness of the Montanide ISA-206, ISA-70 vaccines than alum adjuvant vaccine that reduce the incidence and severity of clinical cases of mastitis. Hence, further studies should be done to elucidate the possibility of field application.