There is a need for plasma-based tests that can directly measure the extent of β-cell injury in vivo in patients receiving islet grafts and in animal models. In this study, we propose protein phosphatase 1, regulatory (inhibitor) subunit 1A (PPP1R1A) as a novel biomarker for acute β-cell destruction. Liquid chromatography–tandem mass spectrometry proteome analysis of fluorescence-activated cell sorter–purified β-cells, tissue-comparative Western blotting, and immunohistochemistry indicated relatively high molar abundance and selectivity of PPP1R1A in β-cells. PPP1R1A was discharged into the extracellular space of chemically injured rat and human islets in vitro, proportionate to the extent of β-cell death. Streptozotocin injection in rats led to a progressive PPP1R1A depletion from the cytoplasm of disintegrating β-cells and a marked surge in plasma levels detectable by an affinity-capture method. A similar massive PPP1R1A discharge in blood was also detected in three patients immediately after intraportal islet transplantation. Our findings provide first proof-of-principle for PPP1R1A as real-time biomarker of β-cell destruction in animal models and patients and warrant development of more sensitive methods for its further validation in clinical trials.