A new strategy for quantitative analysis of a major clinical biochemical indicator called glycated hemoglobin (HbA1c) was proposed. The technique was based on the simultaneous near-infrared (NIR) spectral determination of hemoglobin (Hb) and absolute HbA1c content (Hb · HbA1c) in human hemolysate samples. Wavelength selections were accomplished using the improved moving window partial least square (MWPLS) method for stability. Each model was established using an approach based on randomness, similarity, and stability to obtain objective, stable, and practical models. The optimal wavebands obtained using MWPLS were 958 to 1036nm for Hb and 1492 to 1858 nm for Hb · HbA1c, which were within the NIR overtone region. The validation root mean square error and validation correlation coefficients of prediction (V -SEP, V -R_P) were 3.4 g L^-1 and 0.967 for Hb, respectively, whereas the corresponding values for Hb · HbA1c were 0.63 g L^-1 and 0.913. The corresponding V -SEP and V -R_P were 0.40% and 0.829 for the relative percentage of HbA1c. The experimental results confirm the feasibility for the quantification of HbA1c based on simultaneous NIR spectroscopic analyses of Hb and Hb · HbA1c.