Aim: To explore the interference of a hemoglobin E on glycated hemoglobin (HbA1c) measurement on two methodologies including high performance liquid chromatography (HPLC) and immunoassay. Background: HbA1c is an invincible analyte in modern laboratory medicine era. The role of HbA1c in diabetes mellitus has expanded from prognosis to diagnosis of diabetes. Hence, it becomes an essential responsibility of the medical testing laboratories to deliver quality results to the patients. The quality of a result is determined by several factors. One essential determinant of the quality of an analyte in a clinical laboratory is the choice of method of testing. HbA1c has various standardized methods of measurement, each of it having its own advantages and limitations. One significant limitation is interference from abnormal hemoglobins. Case description: In our case report, we have tried to explore the interference of hemoglobin E on HbA1c measurement on two methodologies including HPLC and immunoassay. In our case scenario, HbA1c immunoassay could produce a reliable HbA1c result, while HPLC was significantly being interfered by the hemoglobin variant. Conclusion: With respect to HbA1c, each method has its own advantages and limitations. It is the responsibility of the laboratories to understand, adopt, and optimally utilize these methods based on the needs. Clinical significance: HbA1c is the gold standard investigation for monitoring patients with diabetes mellitus. National Glycohemoglobin Standardization Program (NGSP) provides traceability and improvement in standardization of methods used for A1C measurement. Each method has its own limitations; the most significant from the clinical significance point of view is being interfered by the abnormal hemoglobins. Hence, each laboratory must understand the strengths and limitations of the available methods in their premises and hold the responsibility for reporting clinically relevant results to its patients.