Aims: Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. Methods: We developed (pro)insulin antigens with alternative placements of a NanoLuc™ luciferase reporter (NLuc). Performance in LIPS was evaluated by testing sera from new onset T1D (n = 80), blood donors (n = 123), schoolchildren (n = 186), first-degree relatives (FDRs) from the Bart’s Oxford family study (n = 53) and from the Belgian Diabetes Registry (n = 136), coded sera from the Islet Autoantibody Standardization Program (IASP) (T1D n = 50, blood donors n = 90). Results: IAA LIPS based on B chain-NLuc proinsulin or B chain-NLuc insulin, in which NLuc was fused at the C-terminus of the insulin B chain, required only 2 μL of serum and a short incubation time, showed high concordance with RBA (Spearman r = 0.866 and 0.833, respectively), high assay performance (B chain-NLuc proinsulin ROC-AUC = 0.894 and B chain-NLuc insulin ROC-AUC = 0.916), and an adjusted sensitivity at 95% specificity ranking on par with the best assays submitted to the two most recent IASP workshops. In FDRs, the IAA LIPS showed improved discrimination of progressors to T1D compared to RBA. Conclusions: We established a novel high-performance non-radioactive IAA LIPS that might replace the current gold standard RBA and find wide application in the study of the IAA response in T1D.