Assisted reproduction technologies are essential for propagating endangered wild felids. Artificial insemination (AI) has been reported in several wild feline species, but pregnancy rates are low, partially owing to failures of current hormonal stimulation protocols. Therefore, this study describes the application of reliable methods to monitor ovarian activity and the development of an effective hormonal protocol to induce oestrus and ovulation in African lions. Application of porcine FSH and porcine LH was shown to be effective for inducing follicular growth and ovulation, and this regimen appeared to be superior to protocols described earlier in terms of ovulation and fertilization rates. Furthermore, non-surgical AI was performed successfully in lions, and uterine-stage embryos were collected and cryopreserved. African lions may serve as a valuable model to develop assisted reproduction for propagation of relic zoo populations in the critically endangered Asian lion or Barbary lion.