Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito-and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/ complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants. Keywords Neurotoxicity • Mitotoxicity • Metabolic reprogramming • High-throughput toxicity screening • High content imaging • Mechanistic safety assessment Abbreviations ADP Adenosine triphosphate AOP Adverse outcome pathway Asp l-Aspartate ATP Adenosine diphosphate cAMP N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate cI-V MRC complex I-V CNS Central nervous system Cyt c Cytochrome c FAD(H 2) Flavin adenine dinucleotide (FAD: oxidized, FADH2: reduced) FCCP Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone G6P Glucose-6-phosphate Gal1P Galactose-1-phosphate GALK Galactokinase GALT Galactose-1-phosphate uridylyltransferase GDNF Glial derived neurotrophic factor Glc1P Glucose-1-phosphate Hex Hexose, in this study either glucose or galactose HK Hexokinase Lac Lactate Mal Malate MoA Mode of action MPP 1-Methyl-4-phenylpyridinium Electronic supplementary material The online version of this article (