A validated spectrophotometric method has been developed for the determination of uranyl ion in soil samples. The method is based on the complexation reaction between uranyl ion and rifampicin in methanol-water medium at room temperature. The method is followed spectrophotometrically by measuring the absorbance at 375 nm. Under the optimized experimental conditions, Beer's law is obeyed in the concentration range of 1.35-20.25 mg mL -1 with apparent molar absorptivity and Sandell's sensitivity of 8.0 × 10 3 L mol -1 cm -1 and 0.042 mg/cm 2 /0.001 absorbance unit, respectively. The interference of a large number of anions and cations has been investigated and the optimized conditions developed have been utilized for the determination of uranium(VI) in soil samples. The three sigma detection limit (n = 9) for uranyl ion was found to be 0.20 mg mL -1 . The proposed method was successfully applied to the determination of uranyl ion in soil samples.