A preventative vaccine against human cytomegalovirus (HCMV) infection and disease remains an unmet medical need. Several attenuated virus and antigen-based HCMV vaccine candidates have been proposed; however, development challenges have limited their progression through the clinical pipeline. A high-throughput and robust relative potency assay, Imaging of Relative Viral Expression (IRVE), was developed and applied to measure the infection of a live-attenuated HCMV vaccine candidate in ARPE-19 epithelial cells. The IRVE assay measures HCMV infection by immunostaining Immediate Early 1 (IE1) protein and enumeration of IE1-positive, infected cells against total cells. Increased throughput was accomplished using 384-well plate automation on a custom-designed integrated robotic system. The IRVE assay effectively measures relative potency changes in an HCMV vaccine candidate under different upstream processes, downstream processes, and formulation conditions. Key assay parameters including microplate format, cell density, serum concentration, infection time and influence of cell age were evaluated and optimized. The IRVE assay was correlated to historical, lower throughput HCMV potency assays, including plaque and Infectivity of Early Gene Expression (IEE), validating its application as a potency screening tool. The IRVE assay has been successfully implemented to support HCMV vaccine development over several years of clinical development.