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The evolution of blood glucose assays reflects the constant progress in laboratory medicine toward increasing analytical precision, serving both diagnosis and patient management. While pioneering, early chemical methods had significant limitations in terms of specificity and reproducibility. Their gradual replacement by enzymatic techniques, particularly those based on glucose oxidase and later on hexokinase, represented a major advance, improving the reliability, sensitivity, and standardization of assays. These developments led to the establishment of internationally recognized reference values and diagnostic thresholds. However, the question of their comparability with the analytical methods currently used in laboratories remains. In this context, this review aims to trace the historical evolution of blood glucose assays and their associated reference values. This involves addressing, on the one hand, reference values according to sampling conditions (fasting, postprandial, oral glucose tolerance test), and on the other hand, diagnostic thresholds for diabetes mellitus. These values are based on recommendations from international scientific organizations, such as the World Health Organization and the American Diabetes Association.