Cardiometabolic syndrome is profoundly influenced by pathways that lie at the interface of metabolism, inflammation and stress and activated in obesity. Metabolic overload initiates a low-grade, chronic inflammatory response, known as metaflammation and which promotes the complications of Cardiometabolic Syndrome. However, the molecular mechanisms linking metabolic stress to immune activation remain elusive. Earlier studies demonstrated metabolic overload leads to endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR is essentially an adaptive signaling emanating from the ER to cope with cellular stress. In addition to its protective responses and stimulation of ER biogenesis, the UPR can also impinge on inflammatory signaling. Prolonged ER stress is detrimental for cells and contributes to obesity, diabetes and atherosclerosis in part by activating pro-inflammatory and pro-apoptotic pathways. Moreover, the existence of inflammatory mediators, chemotactic cytokines, reactive oxygen species and other detrimental signals in obesity make it even more challenging for the ER to establish and maintain a healthy metabolic equilibrium. Clarifying the regulatory mechanisms and novel players in a metabolically driven ER stress response is needed to develop specific and effective new therapeutic strategies for cardiometabolic syndrome. Our research group investigates the UPR that can sense the excess of nutrients and couple to inflammation, and whether its unique operation under metabolic stress can be suitable for therapeutic exploitation in cardiometabolic diseases. A major goal of our studies is to elucidate the molecular differences between metabolic ER stress and the adaptive UPR that could be therapeutically exploited in cardiometabolic syndrome. We adapted a chemical genetic approach to probe the full range of substrates of the proximal UPR kinases, inositol-requiring enzyme1 (IRE1) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), during metabolic stress. This method, which yields a monospecific inhibitor or activator of a kinase involves targeting of a conserved “gatekeeper” amino acid residue in the ATP binding pocket to a small amino acid, enlarging the pocket sufficient enough to accommodate bulky ATP analogs that act as inhibitor or activator. By coupling this to proteomics we are identifying novel substrates for IRE1 and PERK, which may become novel and specific therapeutic targets to modify metabolic ER stress. Moreover, using the same approach in vivo to specifically modify IRE1 and PERK kinase activity during atherogenesis, we are investigating IRE1 and PERK’s direct contributions to inflammation, lipid metabolism and atherosclerosis.