Nuclear pore complexes are pathways for nuclear-cytoplasmic communication that participate in chromatin organization. Here, we present a protocol to image and quantify the number of nuclear pore complexes in cells. We describe steps for cell plating and culture, immunofluorescence detection, and confocal microscopy visualization of nuclear pore complexes. We then detail quantification and 3D data analysis. This protocol utilizes digital thresholding under human supervision for quantification of nuclear pore complexes. For complete details on the use and execution of this protocol, please refer to Han et al.1.