FSH plays an essential role in regulating the crucial functions of male and female reproductive systems. It is widely applied in the treatment of an ovulatory infertility and low gonadotropin-induced hypogonadism (HH). Considering the rapidly growing market of FSH biotherapeutics and the importance of its biological activity, it is urgent to explore more robust bioassay to evaluate the FSH potency. The assay based upon FSH-induced increases in ovarian weight from HCG-treated immature rats is considered the only standard assay to assess the FSH potency. However, the assay has certain drawbacks, such as complicated procedures, long assay period (4 days), high variability, and sacrifice of the experimental animals after treatment. Owing to the large coefficient of variation in animal experiments, the release of hundreds of batches of products per year will cost tens of thousands of rats, which is inconsistent with the 3R principle of animal protection and animal welfare.