Cell volume is a critical parameter for the biology of living species and has an impact on virtually all cellular functions. In Schizosaccharomyces pombe, the regulation of cell size has been the focus of intense investigation, and mechanisms that couple size control with cell cycle progression have been identified. In fission yeast, cell length at division is generally used as a proxy for determining cell size. However, it only allows for an inaccurate evaluation of this critical parameter and neglects potential changes in cell morphology and diameter, which can strongly impact cell volume. Until recently, one of the major obstacles for studying the complexity of cell size regulation in fission yeast has been the lack of a robust method for high-throughput, direct measurement of single-cell volume. Here, we provide a comprehensive protocol for S. pombe cell volume determination based on the Fluorescence eXclusion method and microfluidics technologies. This approach makes it possible to reliably describe cell volume and its distribution in populations of fission yeast cells.