The mammalian oocytes are important materials for study of meiotic cell cycle in developmental biology and cell biology. Confocal microscopy is a powerful technique in detecting the spindle-associated proteins in oocytes. However, in order to obtain the ideal results, some technological modifications should be made according to the characteristics of mammalian oocytes. Among them, the oocyte preparation, fixation, membrane permeabilization, fat extraction, and antibody incubation are the key steps. Furthermore, the differences among oocytes from various mammalian species should be considered in the actual manipulation of confocal microscopy.