Vaccine potency is a critical quality attribute to ensure vaccine’s safety and efficacy. The standard potency assay for vaccines is the traditional cell culture infectious dose 50 % (CCID50). However, this method often yields highly variable results, presenting low sensitivity and requiring a lengthy analysis. Here, we report a flow cytometry-based method for potency assessment of the Rotarix rotavirus vaccine. This method quantifies infected cells by detecting VP7 viral protein in MA104 cells, integrating fluorescence-automated cell counting with a high-throughput system. Determination of adequate cell seeding timepoint, target cell concentration and endpoint after infection are key parameters for the precision, robustness, and accuracy of titer determination. We established optimized assay conditions for lyophilized and final vaccine formulations. Data from both workflows are in line with the CCID50 range of concentrations. The coefficient of variation for inter- and intra-assay for both formulations is ≈ 2 %. Moreover, the flow cytometry method reduced the hands-on and analysis time by at least 4 days, offering a rapid high-throughput alternative tool to streamline the challenges associated with potency testing for viral-based vaccines, impacting therapeutics time to market.