Nanoparticles (NP) often interfere with the mechanism and interpretation of high throughput in vitro toxicity assays. This interference may occur at any time during the assay and spans most NP systems. This study reports on a specific type of gold NP assay interference, where unmodified gold NPs were able to traffic certain assay molecules that contained primary amines across the cell membrane resulting in false positive results for toxicity assays. The enhanced assay molecule permeability was eliminated when the gold NP surface was both sterically and chemically blocked by polyethylene glycol (PEG). The results support the growing consensus that appropriate controls and assay validation should occur prior to interpretation of results of assays using NP.