Methylglyoxal (MG), a reactive metabolic byproduct and a precursor of advanced glycation endproducts (AGEs), is elevated in diabetes. In the body MG is free or reversibly or irreversibly bound (mostly with proteins). Variable plasma MG values have been reported. MG is commonly measured using high performance liquid chromatography. We tested several protocols on different biological samples, which resulted in significant differences in MG values measured in a given sample. The different values do not appear due to the release and detection of bound MG under assay conditions. Protocols that provide consistent values of MG in biological samples are recommended.