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Human-derived three-dimensional (3D) in vitro models are advanced human cell-based model for their complexity, relevance and application in toxicity testing. Intracellular accumulation of methylglyoxal (MGO), the most potent glycating agent in humans, mainly generated as a by-product of glycolysis, is associated with age-related diseases including neurodegenerative disorders.In our study, 3D human stem-cell-derived neuronal spheroids were set up and applied to evaluate cytotoxic effects after short-term (5 to 48 h) treatments with different MGO concentrations, including low levels, taking into consideration several biochemical endpoints.In MGO-treated neurospheroids, reduced cell growth proliferation and decreased cell viability occurred early from 5-10 μM, and their compactness diminished starting from 100 μM, apparently without affecting spheroid size. MGO markedly caused loss of the neuronal markers MAP-2 and NSE from 10-50 μM, decreased the detoxifying Glo1 enzyme from 50 μM, and activated NF-kB by nuclear translocation.The cytochemical evaluation of the 3D sections showed the presence of necrotic cells with loss of nuclei. Apoptotic cells were observed from 50 μM MGO after 48 h, and from 100 μM after 24 h. MGO (50–10 µM) also induced modifications of the cell–cell and cell-ECM interactions. These effects worsened at the higher concentrations (300–500 µM).In 3D neuronal spheroids, MGO tested concentrations comparable to human samples levels measured in MGO-associated diseases, altered neuronal key signalling endpoints relevant for the pathogenesis of neurodegenerative diseases and aging. The findings also demonstrated that the use of 3D neuronal spheroids of human origin can be useful in a strategy in vitro for testing MGO and other dicarbonyls evaluation.