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Fluorescence-based techniques are prevalent in studies of nanomedicine-targeting to cells and tissues. However, fluorescence-based studies are rarely quantitative, thus prohibiting direct comparisons of nanomedicine-performance across studies. With this Commentary , we aim to provoke critical thinking about experimental design by treating some often-overlooked pitfalls in 'quantitative' fluorescence-based experimentation. Focusing on fluorescence-labeled nanoparticles, we cover mechanisms like solvent-interactions and fluorophore-dissociation, which disqualify the assumption that ' a higher fluorescence readout ' translates directly to ' a better targeting efficacy '. With departure in recent literature, we propose guidelines for circumventing these pitfalls in studies of tissue-accumulation and cell-uptake, thus covering fluorescence-based techniques like bulk solution fluorescence measurements, fluorescence microscopy, flow cytometry, and infrared fluorescence imaging. With this, we hope to lay a foundation for more 'quantitative thinking' during experimental design, enabling (for example) the estimation and reporting of actual numbers of fluorescent nanoparticles accumulated in cells and organs. [Display omitted] • Stresses the importance of more quantitative approaches in fluorescence-based nanomedicine studies • Provides a brief introduction to some fundamental principles of fluorescence in simple language • Treats fluorescence-based measurements in bulk solution, microscopy, flow cytometry and tissue/body imaging • Highlights often-overlooked challenges related to interactions between fluorophores and the biological environment • Presents strategies toward counting actual numbers of nanoparticles in cells and organs [ABSTRACT FROM AUTHOR]