Background: Plasma proinsulin concentrations are used diagnostically to investigate hypoglycemia. They have also been proposed as a marker of β-cell function, particularly as a ratio with C-peptide. Immunoassays remain the primary method for measuring proinsulin despite potential limitations. Mass spectrometry-based assays have been described, but are semiquantitative or rely on nano-flow liquid chromatography, which is not suitable for routine clinical use. We aimed to develop a normal-flow liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) to quantify proinsulin and its partially processed forms in human plasma and ensure accuracy over time with distributable well-characterized calibrators. Methods: Sample preparation consists of protein precipitation, Glu-C digestion, peptide immunoaffinity enrichment, and LC-MS/MS analysis of two surrogate peptides. The peptide RGFFYTPKTRREAE spans the cleavage site for des-31,32-proinsulin and GSLQKRGIVE spans the site for des-64,65-proinsulin. A purified protein calibration material was characterized and used to value-assign a matrix-matched single-point calibration material. Results: The assay demonstrated good precision and linearity, with a lower limit of the measuring interval of 2.8 pM for RGFFYTPKTRREAE and 8.8 pM for GSLQKRGIVE. The concentration des-31,32-proinsulin increased more during childhood than intact proinsulin. Participants with type 1 diabetes-associated autoantibodies had higher proinsulin-to-C-peptide ratios. Method comparison with two commercial immunoassays revealed variable cross-reactivity with insulin. Conclusion: The validated assay is robust and will be a useful tool for advancing studies of β-cell function. A detailed standard operating procedure, well-characterized calibration material, and monoclonal antibodies are available to facilitate adoption in other laboratories.