Mitotic proteins are well characterized and their cell cycle regulation roles studied extensively. Specific mitotic proteins can be key targets for controlling de regulated cell cycles. In the present study, cytosolic proteins of CHO (Chinese Hamster Ovary) cells were isolated and used for generating a range of monoclonal antibodies. Of the two antigenic doses utilized, 20 microg and 15 microg doses gave fusion efficiencies of 52.1% and 31.5% respectively. The specific efficiencies were found to be 24.5% for the 20 microg antigenic dose and was 20.45% for the 15 microg dose. Further screening showed 20 MAbs to common mitotic and interphase proteins, 5 specific to unique mitotic proteins and 3 to unique interphase proteins. MPFs do not exhibit species barriers and induce chromatin condensation and act as M- phase check point control molecules. Monoclonal antibodies to specific mitotic proteins can be very useful for various applications such as imaging tools, as possible mitotic inhibitors and also for affinity purification of specific proteins of interest. Antibodies specific to common mitotic and interphase proteins can also be of similar importance. Whole extract was used as immunogen also to present the mitotic proteins much better than isolated proteins.