Despite postulated potential for wildlife conservation, success of assisted reproduction techniques (ART) in ex-situ feline breeding remains <25%. The aim of this project was to develop a simplified, non-surgical artificial insemination (AI) protocol for African lions (Panthera leo), using an exogenous GnRH analogue to induce ovulation in females presenting natural oestrus, and minimizing manipulation of the animals. Four protocols were tested in five trained lionesses (3.5–8 years), for a total of 14 inseminations (2–4 per lioness). These protocols differed in the time lapse between GnRH injection and insemination, on days 4, 5, or 6 from onset of natural oestrus, determined by daily behavioural observation and vaginal cytology. Semen was collected from 8 different males by urethral catheterization and electro-ejaculation, during full anaesthesia. Females were immediately immobilized for AI after semen collection. After transrectal ultrasound examination of the reproductive tract, insemination was performed either intravaginal or transcervical using a commercial dog urinary catheter (2.0 × 500 mm, Buster®, Krusse, South Africa) with a metal stylet. A single intramuscular dose of exogenous GnRH (20 μg burserelin-acetate, Receptal®, MSD, Intervet, South Africa) administered 30 or 48 h before AI or during the AI procedure induced ovulation successfully, as all females entered either a non-pregnant luteal phase of 59.6 ± 0.95 days (n = 10) or a pregnant luteal phase of 111.7 ± 0.33 days (n = 3). However, the timespan between GnRH injection and end of behavioural and/or cytological oestrus differed widely (range: 0–120 h). The final pregnancy success rate was 33.3%.