Objectives:Hemoglobin (Hb) variants remain an important cause of erroneous HbA1c results. We present an approach to overcome the interference of Hb variants on HbA1c measurements using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods:Samples containing or not containing Hb variants were analyzed for HbA1c using an MALDI-TOF MS system (QuanTOF) and a boronate affinity comparative method (Ultra2). For QuanTOF, two sets of HbA1c values were obtained through α- and β-chain glycation. Results:A robust correlation between the glycation degrees of the α- and β-chains was found, and HbA1c values derived from α- and β-chain glycation correlated well with the Ultra2 results. Statistically significant differences (p<0.01) were found for all the Hb variants tested. When using the conventional β-chain glycation to determine HbA1c, clinically significant differences were only found among samples containing β-chain variants detected by QuanTOF (i.e., Hb J-Bangkok, Hb G-Coushatta, and Hb G-Taipei). In contrast, based on α-chain glycation, no clinically significant differences were found for these three variants. Conclusions:In addition to conventional β-chain glycation, α-chain glycation can be used to calculate HbA1c values. The interference of Hb variants on HbA1c quantification can be overcome by employing the glycation of the globin chain without a genetic variant to estimate HbA1c values.