The aquaculture of meagre (Argyrosomus regius) requires methods for the control of reproduction that enable the production of families from specific individuals for selective breeding programs. We experimentally determined the parameters required for an in vitro fertilisation protocol. A total of 14 females and 5 males (mean ± S.D. weights of 20.45 ± 6.22 and 15.94 ± 2.75 kg, respectively) were used. Selected females had vitellogenic oocytes >550 μm in diameter and males had fluid sperm upon application of abdominal pressure. Both sexes were treated with an injection of 15 μg kg−1 of gonadotropin-releasing hormone agonist (GnRHa) to induce oocyte maturation/ovulation and enhance sperm production. To determine the timing of ovulation and window of high egg viability, females were stripped serially every 2.5 h beginning 35 h after GnRHa treatment. Sperm was obtained 24 h after GnRHa treatment and was diluted 1/4 in modified Leibovitz for storage at 4 °C until use. Sperm quality parameters such as percentage initial spermatozoa motility, duration of motility, velocity and density were determined using computer assisted sperm analysis (CASA). In vitro inseminations were made in duplicate or triplicate batches of eggs from each spawn by mixing 0.5–1 mL of eggs, 20–40 μL diluted sperm (pooled from two males) and 100 mL of seawater. Fertilisation success was examined at spermatozoa (spz): egg ratios between ~2000 and 400,000 spz egg−1. The optimal time for stripping ovulated females was ≤3 h after ovulation, which was the window of optimal egg viability. Ovulation under the conditions of this study was close to 38 h after GnRHa treatment, with a range from 35 to 41 h. Beginning from 3 h after ovulation, egg viability declined probably due to overripening. Sperm diluted in Leibovitz maintained motility and velocity for as long as 7 h after collection. Spermatozoa motility (%) and average path velocity (VAP, μm/s) of sperm samples obtained from males before GnRHa injection declined rapidly after activation compared to the samples obtained 24 h post-injection, with significant decreases respectively after 75 and 45 s. A minimum ratio of 150,000 spermatozoa egg−1 was necessary to ensure high fertilisation success. The acquired knowledge of the present study will aid the aquaculture industry and future research on selective breeding programs for meagre.