BackgroundC‐peptide is used widely as a marker of insulin secretion, and it participates in the inflammatory response and contributes to the development of coronary artery disease (CAD) in patients with type 2 diabetes mellitus (T2DM). Previous studies have reported that C‐peptide measurement was unaffected by hemolysis. However, we found that hemolysis negatively affected C‐peptide assay in routine laboratory practice. We further established and validated an individualized hemolysis correction equation to correct and report accurate serum C‐peptide results for hemolyzed samples.MethodsWe studied the effects of hemolysis on C‐peptide assay by adding lysed self red blood cells (self‐RBCs) to serum. An individualized correction equation was derived. Further, we evaluated the performance of this individualized correction equation by artificially hemolyzed samples.ResultsC‐peptide concentration decreased with increasing degree and exposure time of hemolysis. The individualized hemolysis correction equation derived: C‐Pcorr = C‐Pmeas/(0.969−1.5Hbserum/plasma−5.394 ×10−5 Time), which can correct bias in C‐peptide measurement caused by hemolysis.ConclusionsHemolysis negatively affects C‐peptide measurement. We can correct and report accurate serum C‐peptide results for a wide range of degrees of sample hemolysis by individualized hemolysis correction equation for C‐peptide assay. This correction would improve diagnostic accuracy and reduce inappropriate therapeutic decisions.