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Three\u2010dimensional spheroid models can be used to afford greater tissue representation due to their heterogeneous phenotype and complex molecular architecture. This study developed an A549 alveolar carcinoma spheroid model for cytotoxicity assessment and mechanistic evaluation of functionalized AuNPs. A549 spheroids were generated using an agarose micro\u2010mold and were characterized (morphology, acid phosphatase activity, protein content) over 21 culturing days. The 72\u2010h cytotoxicity of carboxyl\u2010polyethylene glycol\u2010 (PCOOH\u2010) and amine\u2010polyethylene glycol\u2010 (PNH<jats:sub>2<\/jats:sub>\u2010) functionalized AuNPs against Day 7 spheroids was assessed by determining spheroid morphology, acid phosphatase activity, protein content, caspase\u20103\/7 activity, and cell cycle kinetics. Spheroids remained stable over the experimental period. Although the A549 spheroids' volume increased while remaining viable over the culturing period, structural integrity decreased from Day 14 onwards. The PCOOH\u2010AuNPs lacked cytotoxicity at a maximum concentration of 1.2\u2009\u00d7\u200910<jats:sup>12<\/jats:sup> nanoparticles\/mL with no prominent alteration to the cellular processes investigated, while the PNH<jats:sub>2<\/jats:sub>\u2010AuNPs (at a maximum of 4.5\u2009\u00d7\u200910<jats:sup>12<\/jats:sup> nanoparticles\/mL) displayed dose\u2010 and time\u2010dependent cytotoxicity with associated loss of spheroid compactness, debris formation, DNA fragmentation, and a 75% reduction in acid phosphatase activity. Differentiation between cytotoxic and non\u2010cytotoxic AuNPs was achieved, with preliminary elucidation of cytotoxicity endpoints. The PNH<jats:sub>2<\/jats:sub>\u2010AuNPs promote cytotoxicity by modulating cellular kinetics while destabilizing the spheroid ultrastructure. 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