Folic acid is the fortificant of choice mainly because of its stability. The mandatory fortification program was implemented based on the evidence that increased intake of folic acid in the periconceptional period reduced the prevalence of neural tube defects affected pregnancies. This study aimed to determine the transport of folic acid from fortified bread across Caco-2 cells, and to compare this with the transport of 5-methyltetrahydrofolate (5-MTHF). Optimised in vitro digestion involved the addition of ascorbic acid at physiological concentration to preserve folate. Quantitative analyses were performed using the previously developed ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method with a slight modification to the UPLC gradient. Our study found that in the in vitro digested bread, the measured 5-MTHF concentrations were significantly higher in the presence of ascorbic acid than in its absence. The transport of folic acid across Caco-2 cells from fortified bread was approximately 14% and not significantly different from the transport of folic acid solution. The experimental data also revealed that transport of 5-MTHF from fortified bread was approximately 1.7%, whilst in control experiments 5% of 5-MTHF standard solution was transported across the cells. The results of the current study suggest that transport of folic acid across Caco-2 cells was greater than transport of 5-MTHF.