A general strategy to carry out cell uptake and biodistribution studies is to label nanoparticles (NPs) with a fluorescent dye. However, the comparative study of different dye-loaded NPs remains difficult owing to uncontrolled dye quenching and de-quenching. Here we compared two types of dye-labeled NPs and demonstrated their distinct properties. NPs with dye molecules at a solid state suffer from dye quenching, so the dye release and/or NP degradation in biological environments leads to a several-fold increase of fluorescence intensity despite the same amount of NPs, owing to the state switch from quenching to de-quenching. In contrast, NPs with dye molecules at a soluble state exhibit no quenching effect. To standardize the comparative study, we propose two possible solutions: using lower dye loading or using medium analysis for quantifying cell uptake of NPs. This work provides valuable insights into selecting valid quantification methods for bio-nano studies.