The endoplasmic reticulum has been likened to the production and packaging department of a manufacturing plant, an action-packed synthesis area that distributes manufactured things to the rest of the cell. The ER serves as a multifaceted gatekeeper of the secretory biosynthetic pathway. Endoplasmic reticulum is a considerable network of intracellular network, Major functions of the ER are a protein folding, proteosynthesis, and post-transcriptional modifications, lipid anabolism and sorting of the protein. Dehydration stress is the most important environmental stress factor, which is seriously affect the chickpea growth, development, and yield production. ER isolation is a very challenging because ER biochemical properties are mostly like the other cellular endomembrane. Most previously published protocols for endoplasmic reticulum isolation are mainly based on the density gradient centrifugation. Although it is a sub optional resolving power. Here in this article, we optimised the endoplasmic reticulum isolation protocol from chickpea tissue. Combined with the physiological analysis, enzymatic analysis, and proteomics analysis, this data will facilitate our better understanding towards the ER proteomics study in chickpea plants during dehydration stress. Generally, proteomics analysis is a powerful tool for the performed at the large scale while the presently enabling also it is a high efficiency procedure. Additionally, in this article we summarize the result of the enzymatic analysis for the purity assessment of the ER, physiological analysis, and proteomics study during the dehydration stress that induce ER stress response. These studies in chickpea plant will facilitate the development of the dehydration stress resistant chickpea in the future.