Uranium determination in environmental samples is faced with problems due to presence of iron and other major elements. Iron is also used many a times for preconcentration of uranium and actinides. Separation of milligram quantity of Fe from microgram quantity of uranium becomes essential during the estimation step. A simple two step procedure has been standardized for separating uranium and iron using anion exchange in 0.025 M H2SO4. Quantitative recovery of uranium was obtained as well as good separation from iron. This method was applied for estimation of uranium in water samples.