To determine the potency of the inactivated rotavirus vaccine (IRV), we developed an enzyme immunoassay (EIA) using a biotin-conjugated RV VP7-specific monoclonal antibody. RV VP7, a pivotal structural protein in the outer capsid layer, governs RV G genotypes and prompts host immune responses, including neutralizing antibodies. This EIA showed high specificity, good linearity, high precision, and high accuracy, with a low limit of detection (LOD) and a limit of quantitation (LOQ) of 0.037 µg/ml RV antigen. The EIA was evaluated and proved suitable for establishing the long-term stability of IRV drug substance (DS) and aluminum-formulated drug product (DP) when stored at -70±10°C and 5±3 °C, respectively. Our results support the use of this EIA to examine the stability and determine the potency, antigen dose, lot-to-lot consistency, and lot release of IRV products. This RV potency assay may serve as an alternative to in vivo potency tests, making it suitable for quality control tests of cGMP IRV lots in clinical trials.