Avocado has a singular synchronous protogynous dichogamy breeding system that promotes outcrossing. In this work different steps have been optimized to improve controlled pollinations in avocado in order to perform basic studies of reproductive biology and directed crosses in breeding programs. The results show that, in order to achieve successful fruit set, male flowers should be collected when all the anthers have dehisced and the pollen transferred by direct contact of the anthers with the stigma during the female stage using preferentially determinate inflorescences and pollinating at least 11 flowers per inflorescence. To perform crosses between cultivars of complementary floral groups, the flowers should be stored under high relative humidity during the hand pollination process. When the crosses involved cultivars of the same floral group, the male flowers should be collected just before anther dehiscence and could be stored for several days at 4 ◦ C. Pollen application should be performed preferentially on flowers within determinate inflorescences or within indeterminate inflorescences where vegetative growth has been removed. The results obtained are useful to optimize controlled pollinations in avocado and can help to increase fruit set in breeding programs and avoid the incorrect interpretation from pollination experiments in studies of reproductive biology.