Abstract: This study employs homogeneous time-resolved fluoroimmunoassay to quantitatively determine the concentration of cyclic adenosine monophosphate (cAMP) within LHCGR(-Ex10)-CRE-luc-HEK293 cells, utilizing a competitive immunoassay method. The research sets forth an in vitro biological activity assessment methodology for luteinizing hormone (LH). Key parameters of the methodology are optimized, and the relative accuracy, intermediate precision, linearity, and range are validated. The optimized experimental conditions are defined as follows: an initial concentration of 200 nmol·mL^-1, a seven-fold gradient dilution, a cell density of 24 000 cells·well^-1, and a duration of incubation of 30 min. The findings indicate that the method exhibits excellent linearity across a relative potency range of 64% to 156%, high relative accuracy, and satisfactory intermediate precision. The successful establishment and validation of a homogeneous time-resolved fluoroimmunoassay for the evaluation of LH biological activity is reported. This methodology is applicable for the assessment of biological activity and quality control of recombinant LH-like drugs, as well as for the differentiation of recombinant LH.