In this study, we examined the usefulness of a simpler and more feasible method for determining the optimal timing of artificial insemination and the conditions for its success in six female common bottlenose dolphins. Pregnancy was successfully achieved in five dolphins by performing intrauterine insemination, using chilled semen stored for less than 3 days or frozen semen within 24 hr of exhibiting a peak serum estradiol (E 2 ) level of 100 pg/mL or higher or on the day with a serum E 2 level of approximately 100 pg/mL, measured with a simple measuring device. We concluded that the determining the optimal timing of intrauterine insemination by measuring serum E 2 levels is a simpler and more useful method compared with the conventional approach.