Determination of folates using liquid chromatography-mass spectrometry (LC-MS) was pioneered by Stokes and Webb. This study developed, optimised and validated a UPLC-MS/MS method to quantify the synthetic folic acid (FA) and the naturally occurring 5-methyltetrahydrofolate (5-MTHF) in Australian fortified breads. Optimised sample preparation prior to analysis involved treatments with α-amylase and rat serum, purification and ultra-centrifugation. Analytes were separated on a Waters Acquity HSS T3 column during a 6 minute run. The validated method is linear over the range 0.39-14 μg FA and 10.1-900 ng 5-MTHF per g fresh bread. The absolute recoveries are 90 % and 76 % for FA and 5-MTHF, respectively. Intraday variations show an RSD of 3% for FA and 18 % for 5-MTHF. The assay is rugged, fast, accurate and sensitive, as well as can detect and quantify the low levels of naturally occurring 5-MTHF in wheat breads.