Significance The nuclear pore complex (NPC) is one of the largest protein assemblies in eukaryotes comprising over 500 nucleoporin subunits. The NPC is essential for the transport of biomolecules across the nuclear envelope; however, due to its enormous size, it has been a challenge to characterize its molecular architecture. Herein, we have developed a widely applicable imaging pipeline to determine the absolute nucleoporin abundances in native yeast NPCs. This reveals that the NPC composition dramatically differs between yeast and humans despite overall conservation of individual subunits. We also applied our imaging pipeline to examine yeast mutants revealing remarkable compositional plasticity of NPCs. Our stoichiometry analyses provide an important resource for the generation of high-resolution structure models of the NPC.