The ostrich industry appears to be in a good position for the development of artificial insemination technology. This study was conducted to investigate the effect of semen collection methods on fertility and hatchability and sperm penetration holes of ostrich. A total of 9 males and 27 females, 3-7 years old, were randomly assigned to three treatment groups with 3 males and 9 females for each group. Three semen collecting methods including manual massage method, dummy method and teaser female method were compared for three consecutive months. Results revealed that the use of teaser female or dummy methods for collecting the semen from male ostrich resulted in significant increase (P≤0.05) infertility (%), hatchability from fertilize eggs (%), hatchability from set eggs (%), and sperm penetration holes and significant decrease (P≤0.05) in embryonic mortality (%) compared with manual massage method during the whole period of experiment. Development of the animal friendly methods (teaser female and dummy methods) for collecting semen from ostriches has advanced considerably in recent years. Semen collected by the teaser or dummy methods gave better results for fertility and hatchability rates and sperm penetration holes.