•30nm gold nanoparticles (AuNPs) are stable in low-serum culture medium for 10days.•Focused ion beam milling exposes AuNPs within cellular cross sections.•AuNPs can be individually resolved by helium ion microscopy.•0.5μg/mL AuNPs do not alter metabolism or neurite outgrowth after 10days exposure.•Cross-slide coefficient of variation for controls (Li+ or AuNPs) is less than 10%. Gold nanoparticles (AuNPs) are promising candidates for medical diagnostics and therapeutics, due to their chemical stability, optical properties, and ease of functionalization. Citrate-stabilized reference materials also have potential as negative controls in toxicology studies of other nanoparticles. Here we examine the impact of 30nm particles on the in vitro development of rat-cortex neural progenitor cells (NPCs), which mimic aspects of the developing neurological environment. AuNPs dispersed in a low-serum culture medium initially agglomerated, but then remained stable during a three day incubation period, and agglomerated only slightly during a ten day incubation period, as determined by dynamic light scattering. Transmission electron microscopy indicated the presence of individual nanoparticles at all timepoints examined. Fixed cells were cross-sectioned by ion milling and imaged by scanning electron microscopy and helium-ion microscopy to evaluate particle incorporation. Individual nanoparticles could be resolved inside cross-sectioned cells. AuNPs were incubated with developing NPCs for ten days at concentrations of 0.5μg/mL Au, 0.1μg/mL Au, or 0.05μg/mL Au. Adenosine triphosphate levels, as determined by bioluminescence measurements sensitive to low cell numbers, were not affected by AuNPs and the particles did not interfere with the assay. Multiple endpoints of neurite outgrowth were not altered by AuNPs, in particular, total neurite outgrowth per cell, a sensitive measure of neuronal development. Slide-level comparisons demonstrated the consistent response of NPCs to gold nanoparticles and a positive control chemical, neuroactive lithium. These results indicate that 30nm citrate-stabilized AuNPs could serve as negative-control reference materials for in vitro measurements of neurite outgrowth.