Background: The aim of this study was to compare the performance of Agilent 1100 HPLC analyser using HbA1c kits manufactured by Gordion Diagnostic (Turkey) with that of Premier Hb9210 using the original kits for the measurement of HbA1c in different patient groups. Methods: Subjects were divided into four groups: Group 1 included 140 diabetic and non-diabetic subjects with normal urea and haemoglobin levels; Group 2 included 84 diabetic and non-diabetic subjects with high urea levels; Group 3 included 44 diabetic and non-diabetic subjects with iron deficiency anaemia; and Group 4 included 52 diabetic and non-diabetic subjects with high haemoglobin levels. EP Evaluator Release 8 program was used to evaluate the resultant data. Results: According to the comparison results of the two methods in all groups, there was an excellent correlation between the two methods (R>0.98). Moderate–low correlation was found between increased urea concentration and the difference of the two methods (R= −0.374, p = 0.0005). The difference between the methods was found to be increased with increased urea concentrations. This difference, although statistically significant, was within the permitted limits. The observed correlation between the difference of the two methods and the low and high haemoglobin concentrations was statistically non-significant (R = 0.149, p = 0.3343; R = 0.263, p = 0.0594). Conclusions: We found that Agilent 1100 HbA1c analyser and Gordions’ HbA1c kit comply with the clinical requirements and are suitable for HbA1c analysis at high levels of urea and Hb and low levels of Hb in diabetic and non-diabetic patients.