Single-molecule detection of quantum dot (QD)-tagged proteins located in the cytoplasm or the nucleus presents a significant challenge in live-cell imaging. First, QDs must enter the cell cytoplasm and reach their molecular target but still preserve cell integrity. Second, the fluorescence of individual QDs must be detected in a noisy environment and distinguished from the autofluorescence of intracellular compartments and organelles. Finally, molecular motion in the cytosol is likely to be three-dimensional, compared to two-dimensional diffusion in the membrane. In this protocol, streptavidin-coated QDs (QD-SAVs) are coupled with biotinylated proteins (ideally in a 1:1 molar ratio) in hypertonic medium. The coupled reaction product (QD-P) is then added to live cells (e.g., mammalian HeLa cells) using a cell-loading technique based on the osmotic lysis of pinocytic vesicles. The osmotic lysis of pinocytic vesicles in hypotonic solution does not alter the viability of cultured cells and does not result in lysosomal enzyme release. By comparison with other internalization techniques, such as microinjection, this method is much simpler and more reproducible because all of the cells are simultaneously loaded under the same conditions. It can provide quantitative information on the movement of intracellular biomolecules, enhancing our understanding of complex biological processes such as signal transduction, cell division, or motility.