Labeling nanoparticles with fluorescent dyes is a common approach to investigate their cell uptake and biodistribution, providing valuable information for the preclinical assessment of nanoparticles for drug delivery. However, the underlying assumption that the fluorescence intensity of dye-labeled nanoparticles correlates positively with the amount of nanoparticles taken up by cells might not be valid under some conditions, as it can be affected by many factors including dye dispersion, dye quenching, and material shading. Here we demonstrated that both nanoparticles with hydrophobic dyes encapsulated inside and nanoparticles with hydrophilic dyes conjugated on the particle surface suffer from different degrees of dye quenching, making it challenging for quantitative comparison of cell uptake of different nanoparticles. To address this challenge, we proposed a possible solution for direct comparative studies of dye-labeled nanoparticles. This work provides valuable information for designing and evaluating different nanoparticles for drug delivery applications.