Cellular structures and biomolecular complexes are not simply assemblies of proteins, but are organized with defined numbers of protein molecules in precise locations. Thus, evaluating the spatial localization and numbers of protein molecules is of fundamental importance in understanding cellular structures and functions. The amounts of proteins of interest have conventionally been determined by biochemical methods. However, biochemical measurements based on the population average have limitations: it is sometimes difficult to determine the amounts of insoluble proteins or low expression proteins localized in small portions of the cell. In contrast, microphotometric measurements using fluorescence microscopes enable us to detect the amounts of such proteins in situ in a particular subcellular region. Here, we describe a method to measure the amounts of fluorescently tagged proteins by fluorescence microscopy, and present an example of an application to nuclear pore proteins in the fission yeast Schizosaccharomyces pombe.