Because nanoparticles are promising tools in drug delivery, quantification of their cellular binding and uptake is an emerging question. Therefore, rhodamine B isothiocyanate-labeled silica nanoparticles with different sizes and surface modifications were investigated concerning their uptake in Caco-2 cells. Flow cytometry studies exhibited a size- and time-dependent association for unmodified nanoparticles (50 and 77 nm), whereas larger particles (94 nm) and polyethylene glycol-modified nanoparticles showed no cellular interaction. A second approach dealt with particles with adsorbed propidium iodide (PI) to distinguish between internalized and adsorbed nanoparticles. These particles only give a fluorescence signal when associated with nucleic acids inside the cell, whereas particles adsorbed to the outer cell surface are not detected. PI-labeled nanoparticles (21 nm) showed a time-dependent uptake, exhibiting a signal in the cytoplasm but less in the nucleus. These novel PI-labeled nanoparticles in combination with flow cytometry are innovative tools for the quantification of nanoparticulate uptake.