There is a clinical need for plasma tests for real-time detection of beta cell destruction, as surrogate endpoint in islet transplantation and immunoprevention trials in type 1 diabetes. This study reports on the use of label-free LC-MS/MS proteomics for bottom-up selection of candidate biomarkers. Ubiquitin COOH-terminal hydrolase 1 (UCHL1) was identified as abundant protein in rat and human beta cells, showing promising beta cell-selectivity, and was selected for further validation in standardized toxicity models. In vitro, H2O2-induced necrosis of INS-1 cells and human islets resulted in intracellular UCHL1 depletion and its extracellular discharge. In vivo, streptozotocin progressively depleted UCHL1 from islet cores and in 50% of animals, an associated plasma UCHL1 surge was detected preceding the GAD65 peak. UCHL1 was cleared with a half-life of 20min. Whole-body dynamic planar imaging of (99m)-Technetium-labeled UCHL1 indicated a rapid UCHL1 uptake in the liver and spleen, followed by urinary excretion of mainly proteolytic UCHL1 fragments. We conclude that LC-MS/MS proteomics is a useful tool to prioritize biomarkers for beta cell injury with promising molar abundance. Despite its consistent UCHL1 discharge by damaged beta cells in vitro, its in vivo use might be restrained by its rapid elimination from plasma.Our bottom-up LC-MS/MS proteomics represents a pragmatic approach to identify protein-type biomarkers of pancreatic beta cell injury. UCHL1 successfully passed sequential validation steps of beta cell-selectivity, antigenicity and toxic discharge in vitro. Whole-body dynamic planar imaging of radiolabeled recombinant UCHL1 indicated rapid clearance through the liver, spleen and urinary excretion of proteolytic fragments, likely explaining non-consistent detection in vivo. Integration of kinetic biomarker clearance studies in the a priori selection criteria is recommended before engaging in resource-intensive custom development of sensitive immunoassays for clinical translation.