Diacetyl is a volatile compound from citrate metabolism responsible for the buttery flavor of washed curd cheeses manufactured with positive cultures citrate (Cit +), such as Prato cheese. Diacetyl can be assayed by a colorimetric method, which is laborious, imprecise and not specific since it concomitantly determines acetoin. However, the colorimetric method requires simple equipment and is inexpensive. This method is based on the precipitation of protein and fat with sodium tungstate and sulfuric acid and subsequent reaction of diacetyl and acetoin with creatine and α-naphthol generating a red-colored compound. Diacetyl can also be determined by solid-phase microextraction coupled to gas chromatography-mass spectrometry (SPME-GC/MS), which is a selective, sensitive, precise and accurate method. The objective of this study was to compare both techniques for determining diacetyl in Prato cheese during ripening (210 days). Both methods were suitable to evaluate the changes on diacetyl content during ripening since they were equally able to detect the diacetyl content decrease during the cheese maturation. The colorimetric method detected diacetyl in the range of 80 to 220 μg/g, while the chromatographic method presented results between 2.0 × 10-4 and 1.6 × 10-3 μg/g, showing to be more sensitive. In addition, the colorimetric method does not discriminate acetoin. There was a positive Pearson correlation of 0.85 (p = 0.0036) between the colorimetric and SPME-GC/MS methods.