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The peptide hormone insulin produced by pancreatic β-cells undergoes post-transcriptional processing before secretion. In particular, C-peptide is cleaved from pro-insulin to generate mature insulin. Here, we introduce a C-peptide-mCherry human iPSC line (HMGUi001-A-8). The line was generated by CRISPR/Cas9 mediated heterozygous insertion of the mCherry sequence into exon 3 of the insulin locus. We demonstrate that the line is pluripotent and efficiently differentiates towards pancreatic β-like cells, which localize a red fluorescent C-peptide-mCherry fusion protein in insulin containing granules. Hence, the HMGUi001-A-8 line is a valuable resource to purify derived β-like cells and follow insulin-containing granules in real time.