The reversible phosphorylation of proteins controls most cellular functions. Protein kinases have been popular drug targets, unlike phosphatases, which remain a drug discovery challenge. Guanabenz and Sephin1 are selective inhibitors of the phosphatase regulatory subunit PPP1R15A (R15A) that prolong the benefit of eIF2α phosphorylation, thereby protecting cells from proteostatic defects. In mice, Sephin1 prevents two neurodegenerative diseases, Charcot-Marie-Tooth 1B (CMT-1B) and SOD1-mediated amyotrophic lateral sclerosis (ALS). However, the molecular basis for R15A inhibition is unknown. Here we reconstituted human recombinant eIF2α holophosphatases, R15A-PP1 and R15B-PP1, whose activity depends on both the catalytic subunit PP1 (protein phosphatase 1) and either R15A or R15B. This system enabled the functional characterization of these holophosphatases and revealed that Guanabenz and Sephin1 induced a selective conformational change in R15A, detected by resistance to limited proteolysis. This altered the recruitment of eIF2α, preventing its dephosphorylation. This work demonstrates that regulatory subunits of phosphatases are valid drug targets and provides the molecular rationale to expand this concept to other phosphatases.