Hydralazine is commonly employed as a time-dependent inhibitor of aldehyde oxidase (AO) in human hepatocytes for reaction phenotyping yet profound inter-lot variability in its inhibitory effects has been reported. To date, the exact mechanistic basis for this observation remains unclarified. Recently, it was reported that the time-dependent inhibition of AO by hydralazine could be reversed by glutathione (GSH). The present study integrates these previously independent observations for the first time by investigating whether GSH-dependent reversibility of hydralazine-mediated AO inactivation contributes to this observed variability. The time-dependent inhibition of human liver cytosolic AO elicited by hydralazine was fully reversed by GSH, whereas no protection was observed for erlotinib, a structurally distinct AO time-dependent inhibitor. Consistent with these findings, the apparent inhibitory potency (IC50) of hydralazine in pooled human hepatocytes was intermediate between GSH-free and GSH-supplemented cytosolic systems, while erlotinib exhibited comparable inhibition across all conditions. These results indicate that GSH-reversible AO inactivation by hydralazine contributes to inter-lot inhibition variability in human hepatocytes and highlight a mechanistic limitation of hydralazine as a chemical inhibitor for AO reaction phenotyping in human hepatocytes.