The interaction of nanoparticles with biological cells is an important issue in nanomedicine. The tunable surface functionality of gold nanoparticles (AuNPs) provides appealing design flexibility to ensure specific interactions with cells, and efficient cellular uptake. Here, we demonstrate the first use of laser desorption/ionization mass spectrometry (LDI-MS) to simultaneously screen the uptake of multiple functionalized AuNPs into cells. The cellular uptake of AuNPs with different surface functionality can be readily determined by using the mass-to-charge (m/z) ratios of their surface ligands. These “mass labels”, which can be read out by LDI-MS, allow different AuNPs to be simultaneously identified and quantified. Surface properties (e.g. charge and hydrophobicity) are key determinants that control the interactions of nanomaterials with cells. This technique is also used to investigate how the serum proteins absorbed on AuNPs surface influence AuNPs and cells interactions, leading to the distinct cellular uptake propensities.