Assisted reproduction techniques in birds have been developed for zootechnical purposes and have been adapted for use in conservation of wild bird species. To develop a technique for obtaining follicles in live hens, 5 Rhode Island red hens (Gallus gallus domesticus) were anesthetized, and abdominal ultrasound was performed to confirm the presence of ovarian follicles. A left celiotomy then was performed to obtain follicles in different stages of maturation for in vitro fertilization. The follicles were located by digital exploration, then extracted by isolating each follicle with the index finger of each hand, holding it by the stigma, and then applying slight traction towards the exterior of the coelomic cavity until the follicle separated from the ovary. In total, 18 of 30 (60%) follicles obtained were suitable for in vitro fertilization, but only 3 (16%) were fertilized successfully. All birds recovered from the procedure and remained in good condition postoperatively. Perfecting assisted reproduction technique holds potential benefits for determining sex of embryos by blastomeres sexing, supporting the conservation efforts of avian species, and benefiting research areas, such as genetic and biopharmaceutical research.