To gain insight into the toxicity induced by VX, a mass spectrometry based phosphoproteomics approach was employed to understand the signaling modulated by VX toxicity in piriform cortex region of the rat brain. We have employed isobaric-based TMT labeling and titanium dioxide- based enrichment strategy to identify and quantify the changes that are modulated by VX. We observed a temporal association of changes in the phosphorylation status of proteins over a 24 hour time course in rats exposed to 1x LD50 VX, with the most notable changes by the first measured time point, 1 hour post exposure. These data fell into five main functional classes of proteins directly or indirectly influenced by changes in phosphorylation: 1) Ion channels/transporters, including ATPases, 2) Kinases/Phosphatases, 3) GTPases, 4) Structural related proteins, and 5) Transcriptional regulatory proteins. This study is the first quantitative phosphoproteomics analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain, and aid in the elucidation of novel molecular targets allowing for improved countermeasure development.