Our work provides new insights into the way that ultrasmall AuNPs, ranging from 2 to 15 nm, penetrate and localize within cultured cancer cells, multicellular spheroids, and tumors in vivo. The 2 and 6 nm gold nanoparticles demonstrated unique advantages over larger nanoparticles in terms of tumor uptake and permeability. Accumulation of nanoparticles in the tumor spheroid model occurred in a size-dependent manner, with 2 and 6 nm AuNPs able to penetrate deeply into tumor tissue. In summary, this work showed how nanoparticles can be designed and functionalized to achieve high levels of accumulation in tumor tissue.