Simple, sensitive and reproducible direct and derivative spectrophotometric methods for the determination of iron(II) and uranium(VI) are reported. The method is based on the formation of orange coloured complex of a stoichiometric ratio 2:3 between iron(II) and 2-hydroxy-3- methoxybenzaldehyde- isonicotinoylhydrazone (HMBAINH) and the formation of yellow coloured complex of stoichiometric ratio 1:1 between uranium(VI) and HMBAINH at pH 4.0. The maximum absorbance of [Fe(II)-HMBAINH] complex was measured at 385nm. Under the optical conditions, Beer’s law is obeyed over the range 0.139-1.396 mg mL-1. The molar absorptivity and detection limits are calculated as 2.0x104 L mol-1 cm-1 and 0.013 mg mL-1 respectively. [U(VI)-HMBAINH] complex shows maximum absorbance at 395nm, Beer’s law range, molar absorptivity and detection limits are 1.19 -11.9 mg mL-1, 0.9 x 104 L mol-1 cm-1, and 0.224 mg mL-1 respectively. A simultaneous third order derivative spectrophotometric method of determination of iron(II) and uranium(VI) is also reported. The proposed methods were successfully employed in the determination of iron and uranium in various environmental, biological and ore sample.