There is global interest in cultivating the red alga Asparagopsis taxiformis, due to its efficacy as a potent anti-methanogenic feed supplement. The development of A. taxiformis cultivation is currently hindered by a lack of information around the conditions required to maximise tetraspore release and thus secure a reliable source of young gametophytes for out-planting. We used a five-week, full factorial experiment to quantify the effects of temperature, irradiance and nutrient supplementation (F/8, potassium iodide (KI), arsenic trioxide (As2O3)) on reproductive output under short-day conditions, using a strain of A. taxiformis native to the Great Barrier Reef. Temperature, irradiance and nutrient supplementation played a pivotal role in reproductive output, which was optimised at 22 °C under 10 µmol photons m−2 s−1, with supplementation of culture water with F/8, As2O3 (98 µg L-1) and KI (166 µg L-1). Tetraspore release under these conditions peaked during the third experimental week, with an average of 2,179 ± 818 germinated tetraspores released per tetrasporophyte over the experimental duration. Once tetrasporophytes were removed from inducing conditions, tetrasporogenesis ceased within 12 days. Our recommendations provide a starting point for enhancing productivity in A. taxiformis hatcheries, to generate a reliable supply of young gametophytes for nursery cultivation.