Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown. The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs. hCG effects were examined according to days in culture and hCG stimulation time. As read-out， we analyzed the gene expression of known hCG targets， protein production， and progesterone secretion. Our results show that with a daily medium exchange， the strongest effect was achieved already 4 days after seeding. On day 4， hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6-8 h after hCG， while 24 h of hCG stimulation was needed for late induced genes. Based on our results， we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24 h later.