Rotavirus is the main cause of viral diarrhea among young children. There exist licensed vaccines, but rotavirus still kills nearly half a million children yearly. Live, attenuated vaccines quickly lose potency at room temperature. My host lab is assisting a Seattle NGO callednPATH and the Bill and Melinda Gates Foundation with developing temperature-stabilized, inexpensive vaccines. The current industry standard assay for measuring vaccine potency is time-consuming, subjective, and low throughput. My goal is to develop an objective, inexpensive, automatable, and high-throughput assay of the potency of candidate, next-generation, temperature-stable rotavirus vaccines. To develop this assay, susceptible cells are infected with candidate vaccine-strain viruses manufactured in India and undergoing development at PATH. After cell membrane modification to allow antibody access to cytoplasmic viral proteins, an anti-rotavirus antibody and a fluorescent secondary antibody are used to detect viral protein within infected cells. Experimental samples undergo single-cell flow cytometry analysis of individual cells to differentiate infected and uninfected cells. The vaccine industry-standard potency assay is performed in parallel to directly compare assays. Comparison and titration of polyclonal and monoclonal rotavirus-specific antibodies identified the optimal antibody and concentration for specific intracellular recognition of rotavirus protein. Testing of secondary antibodies and fluorochromes resulted in cell fluorescence characteristics yielding a favorable signal-to-noise ratio without causing false-positive results. A direct relationship between the amount of live rotavirus input and the percentage of infected cells with good linearity and acceptable coefficients of variation was observed. Future work is focused on reducing material costs, comparing the flow cytometry assay to the industry standard, and adapting the assay to additional rotavirus vaccine strains. Based on the above results, flow cytometry is a promising candidate assay for quantifying rotavirus vaccine potency to assist the development of novel live rotavirus vaccine formulations and storage formats.