BACKGROUND: Hemoglobin A1c (HbA1c) is a good marker for the monitoring of glycemic control in patients with diabetes mellitus and various methods are used for determination of HbA1c. We evaluated the analytical performance of ion-exchange high-performance liquid chromatography system, standard mode (G8 SM) and variant mode (G8 VM) of Tosoh HLC-723 G8 (Tosoh Corporation, Japan) and Cobas Integra 800 (Roche Diagnostics, Germany) using new reagent (Tina-quant Hemoglobin A1c Gen.2, Roche Diagnostics) for immunoassay. METHODS: G8 SM, G8 VM, and Cobas Integra 800 were evaluated for linearity, precision, comparison of method, and speed. Variant II Turbo (Bio-Rad Laboratories, USA) was used for comparison test for above three analyzers. RESULTS: Three instruments showed within-run precision (CVs) of 0.52-1.12% in low level and 0.39-0.90% in high level control material. Total run precision (CVs) were in 0.95-1.26% in low level and 0.60-0.94% in high level control material. The linearity was good for the range of 4-15% and comparison with Variant II turbo was excellent with 0.9878-0.9977 of correlation coefficient. Analytical speed was 2 min/1 min in G8 SM, 3.5 min/1.5 min in G8 VM, and 14.7 min/18 sec in Cobas Integra 800, respectively. CONCLUSIONS: HLC-723 G8 SM and VM, and Cobas Integra 800 were showed excellent linearity, precision, and comparison with other instrument. We should consider the characteristics including analytical method and speed in each instrument when clinical laboratory is planning to introduce different equipment for HbA1c determination.