Many applications of nanomedicines depend on the therapeutic gaining access to the interior of cells. As most proteins and nanoparticles are taken up by endocytosis, determining the properties of nanoparticles that govern uptake is essential. In this review, we examine the current approaches for measuring the cellular uptake of nanoparticles and proteins. We explore the techniques distinguishing material internalized by the cell from material bound to the surface, with a particular focus on recent advances in sensor technology. We also highlight the requirements for quantifying internalization and the pitfalls that can limit data analysis. Finally, we explore the importance of understanding recycling of internalized material back to the cell surface, and the methods that can be used to quantify this. Delivering cargo to specific subcellular locations first requires uptake. Robust techniques that can quantify this event are the critical for developing the next generation of smart, targeted, therapeutic nanoparticles. (c) 2020 Published by Elsevier Inc.