Subcellular proteomics include, in its experimental workflow, steps aimed at purifying organelles. The purity of the subcellular fraction should be assessed before mass spectrometry analysis, in order to confidently conclude the presence of associated specific proteoforms, deepening the knowledge of its biological function. In this chapter, a protocol for isolating endoplasmic reticulum (ER) and purity assessment is reported, and it precedes the proteomic analysis through a gel-free/label-free proteomic approach. Dysfunction of quality-control mechanisms of protein metabolism in ER leads to ER stress. Additionally, ER, which is a calcium-storage organelle, is responsible for signaling and homeostatic function, and calcium homeostasis is required for plant tolerance. With such predominant cell functions, effective protocols to fractionate highly purified ER are needed. Here, isolation methods and purity assessments of ER are described. In addition, a gel-free/label-free proteomic approach of ER is presented.