<p>Validamycin A (VMA), an inhibitor of trehalase was applied as a foliar spray in black gram (<i>Vigna mungo</i> L. Hepper) plants imposed to drought stress to intervene the trehalose pathway, which is involved in conferring desiccation tolerance. Untargeted metabolomics study was conducted using GC–MS, with data analyzed by the Metaboloanalyst platform. Enzyme assays confirmed the decreased activity of trehalase and increased accumulation of trehalose to 1.8 fold in drought-stressed VMA treated leaf tissues when compared to that of control (watered). These samples also revealed significantly high starch content (14.29 mgg⁻¹) compared to that of drought stress (9.36 mgg⁻¹). In drought-stressed plants, amylase and acid invertase activities of leaf increased significantly and metabolomic profiling showed enhanced accumulation of glucose (33-fold) and fructose (sevenfold) which are the products of these enzymes. The heat map of drought-stressed VMA treatment revealed a unique accumulation of palmitic acid, 8,10-dioxaheptadecane, 9-octadecenoic acid, stearic acid, N-acetyl glutamic acid, 3-alpha mannobiose, glyceryl glycoside, pentanedioic acid, myristic acid, sorbitol, 1,4-butanediol, arabitol, tagatose and sucrose. Based on Variable Importance in Projection (VIP) score plot, mannose, lyxose, and oxalic acid are selected as marker metabolites linked with drought tolerance due to VMA treatment. Pathway impact analysis revealed that galactose metabolic pathway was the most affected. This study provides a comprehensive understanding of the metabolites associated with trehalose accumulation that can provide strategies to mitigate drought stress in black gram.</p>