Defect engineering proves to be a highly effective approach for introducing additional open metal sites and porosity into metal–organic frameworks (MOFs), thereby enhancing their gas storage, separation, and chemical catalysis capabilities. However, characterizing defective MOFs, which often exhibit nonuniform pores, presents a significant challenge. While probe molecules have been widely utilized to explore the physical and chemical properties of MOF pores, their application has predominantly been limited to gas- or vapor-phase molecules. In this study, we present a novel approach by employing a size-selective fluorescent protein probe to characterize macroporous defects induced by tartaric acid in a zirconium-based MOF, NU-1000. The spatial visualization of defects using a hemoglobin-based fluorescent probe allows for the identification of distinct structural weak points and defect formation mechanisms in NU-1000 crystallites prepared by various methods. In addition to confirming findings from conventional MOF characterization methods, such as gas sorption isotherms and powdered X-ray diffraction analysis, the hemoglobin-based protein probe unveils structural nuances overlooked by many traditional characterization techniques.