We developed an enzymatic colorimetric method for the quantification of α-D-mannose 1-phosphate by adding phosphomannomutase, mannose 6-phosphate isomerase and glucose 6-phosphate isomerase to a conventional glucose 6-phosphate assay using glucose 6-phosphate dehydrogenase. In this method, α-D-mannose 1-phosphate is converted into D-glucose 6-phosphate via D-mannose 6-phosphate and D-fructose 6-phosphate and the resultant D-glucose 6-phosphate is ultimately converted into 6-phosphogluconolactone under concomitant reduction of thio-NAD⁺ to thio-NADH, which can be quantified by its wavelength of 400 nm. This method is not altered by the presence of D-mannose, D-mannosamine, N-acetyl-D-mannosamine, L-mannose, β-1,4-mannobiose, α-1,2-mannobiose, methyl α-D-mannoside or dimethyl sulfoxide and it would be useful in studies involving enzymes such as phosphorylases belonging to glycoside hydrolase family 130, which release α-D-mannose 1-phosphate as the reaction product.