This Letter presents a new method for differentiating the Au nanospheres attached to the cell surface from those being internalized into the cells. We introduced an etching solution based on I2 and KI that can selectively dissolve the Au nanospheres on the cell surface within a short period of time. The advantage of this etchant is its low toxicity to the cells because it is capable of etching away a relatively large amount of Au nanospheres at a low molar concentration. By combining with quantitative elemental analysis, we found that the deposition of Au nanospheres on the surface of cancer cells was highly dependent on the sign of surface charges on the Au nanospheres. In addition, by fitting the uptake data with a kinetic model, we were able to derive the overall and internalization rate constants for Au nanospheres and both of them were found to be governed by the surface charges on Au nanospheres.