Gold nanoparticles (AuNPs) are highly promising candidates as drug delivery agents into cells of interest. We describe for the first time the multiplexed analysis of nanoparticle uptake by cells using mass spectrometry. We demonstrate that the cellular uptake of functionalized gold nanoparticles with cationic or neutral surface ligands can be readily determined using laser desorption/ionization mass spectrometry of cell lysates. The surface ligands have “mass barcodes” that allow different nanoparticles to be simultaneously identified and quantified at levels as low as 30 pmol. Using this method, we find that subtle changes to AuNP surface functionalities can lead to measurable changes in cellular uptake propensities.