Bioavailability refers to that fraction of nutrients which is utilized by the body out of the total indigested amount. Various direct and indirect methods for the determination of bioavailability are available. We determined the bioavailability of iron from fortified wheat flour using both in vitro and in vivo methods. The bioavailability data will be used to make the recommendation for a fortification strategy in Pakistan. The in vitro bioavailability of iron from fortified wheat flour was determined using in vitro enzymatic digestion and fermentation by simulating the condition of the small intestine and colon in the laboratory. Different products were prepared from the fortified ferrous sulfate (FeSO4) and unfortified wheat flour. The total iron of the samples was measured by the wet-digestion method and analyzed on an atomic absorption spectrometer (AAS). To obtain the percentage of iron released, the samples were subjected to pepsin digestion and dialysis. The dialysate was collected at 3, 6, 9, and 12 hours and read on an AAS. The retentates from the above were subjected to the fermentation condition of the colon by inoculating it with human fecal inoculum and incubating it for 24 hours at 37 degrees C under anerobic conditions. The dialysate was collected at 3- and 6-hour intervals and read on an AAS. More iron was released from fortified wheat flour (4.6%), leavened chapati (6.8%), and Nan (15.1%) than from the unfortified control samples. Fermentation and leavening resulted in a better release, which was evident from in vitro digestion results.