Endoplasmic Reticulum (ER) is the site for secretory and transmembrane protein synthesis and maturation. Disrupted ER homeostasis activates a cascade of ER-to-nucleus signaling pathways termed the Unfolded Protein Response (UPR) to alleviate ER stress and restore ER homeostasis. Causal links between ER stress and a variety of human diseases have been suggested. However, as physiological ER stress is subtle and hard to detect relative to pharmacologically induced ER stress, the significance of ER stress and UPR activation in the pathogenesis of these disorders remains controversial. To this end, our lab developed a Phos-tag based SDS-PAGE method to sensitively detect the phosphorylation of UPR sensors (IRE1[ALPHA]/PERK), a direct indicator of ER stress levels and UPR activation. This method was verified and showed to quantitatively monitor UPR activation in cells expressing misfolded proteins. More significantly, this assay revealed delicate changes in IRE1[ALPHA] phosphorylation in mouse tissues under basal or stimulated physiological conditions that could not be visualized with a regular SDS-PAGE gel. Using this powerful tool in a screen for anti-diabetic reagents, we showed that phenformin, an anti-diabetic drug, stimulated the IRE1[ALPHA] and PERK pathways in both AMPK- and ER stress-dependent manners, revealing a novel crosstalk between UPR and metabolic pathways. To further expand our understanding of physiological ER stress, we turned to a model of chronic ER stress and UPR activation. Specifically, we generated a mouse model deficient for SEL1L, a core factor in ER associated degradation (ERAD). Animals with SEL1L deficiency in B cells (SEL1LCD19) exhibited severely impaired B cell development. Unexpectedly, the developmental defect in SEL1L-null B cells was independent of ER stress-mediated cell death, as UPR sensor phosphorylation and chaperone induction were not detected. Additional data supported a model that the phenotypic defect was due to a block in degradation of VpreB, a key component for a critical checkpoint during B cell developmental. In addition, our method, for the first time, showed an uncoupling between the activation status of UPR sensors and downstream targets in B cell to plasma cell differentiation. Taken together, our Phos-tag gel has proven to be a sensitive and reliable tool for investigating physiological ER stress. We believe this method will be critical in further understanding and elucidating the physiological role of mammalian ER quality control system, and may provide insights into future diagnosis for ER-associated conformational diseases.