Updated project metadata. Toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP causes cellular perturbation by modifying non-cholinesterase targets. Here, we identify novel DDVP-modified targets in lysates of a human hepatocyte-like cell line (HepaRG) using a direct LC-MS assay of DDVP exposed lysates or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. Using these strategies, we show that DDVP forms adduct to several proteins important to the cellular metabolic pathways and cellular differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues.